Molecular markers

ABSTRACT

Nucleic acid based methods for detecting the presence of  E. coli  or  Shigella  or related microorganisms in a sample using one or more  E. coli  or  Shigella  species specific nucleotide sequences are disclosed. More particularly the identification of molecules capable of binding or otherwise facilitating abnormal cell growth or abnormal physiology such as found in cancer or cellular instability is described. Further, molecular probes for performing the nucleic-acid based methods and methods of testing and selecting nucleic acid sequences suitable for same are provided. The methods and polynucleotides are useful inter alia in the testing of food and water samples, for testing for genetic and cellular instability, and for testing benign, pre-neoplastic and neoplastic disease in asymptomatic or symptomatic colorectal or gastric cancer patients or those at risk of the aforementioned conditions or those infected by Escherichieae and with other diseases or conditions.

FIELD OF THE INVENTION

The present invention relates generally to nucleic acid based methodsfor detecting the presence of E. coli or Shigella or relatedmicroorganisms in a sample using one or more E. coli or Shigella speciesspecific nucleotide sequences. More particularly, the present inventionpermits the identification of molecules capable of binding or otherwisefacilitating abnormal cell growth or abnormal physiology such as foundin cancer or cellular instability. The present invention furtherprovides molecular probes for performing the nucleic-acid based methodsof the invention and methods of testing and selecting nucleic acidsequences suitable for same. The methods and polynucleotides of thepresent invention are useful inter alia in the testing of food and watersamples, for testing for genetic and cellular instability, and fortesting for benign, pre-neoplastic and neoplastic disease inasymptomatic or symptomatic colorectal or gastric cancer patients orthose at risk of the aforementioned conditions or those infected byEscherichieae and with other diseases or conditions.

BACKGROUND OF THE INVENTION

The identification of bacteria can be carried out using biochemical,cultural, antibody recognition and molecular biological tests (Feng P CS and Hartman P A: Fluorogenic Assays for Immediate Confirmation ofEscherichia coli. 1982. Falkow S, Habermehl K O. ed: Rapid Methods andAutomation in Microbiology and Immunology. Springer-Verlag, Berlin 1985:30–33. AOAC Official Methods of Analysis 1995. Pepper Ill., Gerba C Pand Brendecke J W: Environmental Microbiology. A laboratory Manual.Academic Press 1995.)

Food and Water Hygiene

Biochemical Test and Culture Medium

The most probable number (MPN) is the common method for the detectionand quantitation of E. coli in foods. This method detects E. coli on thebasis of the bacteria's ability to ferment lactose with the evolution ofgas. Other non-E. coli organisms also ferment lactose and, therefore,several selective enrichment steps are required in order to sequentiallyselect for coliform bacteria and E. coli.

This widely used MPN method has several limitations. Many clinical E.coli isolates are lactose negative and thus are not detected using theMPN method. The MPN method requires a minimum of about four days todetermine the absence of E. coli in food products and about seven daysare required to get confirmed results. The growth of some E. coli,including the serotype 0157:H7 strains, is severely inhibited by theselectivity of the EC broth at 45.5° C. and gas production in the MPNmethod is susceptible to interference by high levels of competitororganisms.

More rapid methods for detecting E. coli are needed because of the timeand accuracy limitations of the MPN method. It has been reported that94% to 97% of E. coli strains possess the B-D-glucuronidase that can bedetected by specific hydrolysis of a synthetic substrate,4-methylumbelliferyl-B-D-glucuronide (MUG), to a fluorescent endproduct. When MUG is incorporated into lauryl sulfate tryptose (LST)broth, 10⁷ to 10⁸ CFU/ml of E. coli will yield this fluorescent productwhich can be detected under longwave UV light. However, a number ofenteropathogenic E. coli including serotype 0157:H7 strains, do notpossess the B-D-glucuronidase enzyme, do not exhibit fluorescence inLST-MUG medium, and therefore yield false-negative results using the MUGmethod. In addition, the selectivity of the method is compromised by thefact that some Shigella, Citrobacter, Ecterobacter, Klebsiella,Salmonella, and Yersinia species also produce B-D-glucuronidase andtherefore yield false-positive results.

Another widely used test, the Analytical profile index (API) teststrips, produced by BioMerieux (France), may be used to obtain testresults quickly. These consist of a series of miniature capsules onmolded plastic strips, each of which contains a sterile dehydratedmedium in powder form. Addition of water containing a bacterialsuspension simultaneously re-hydrates and inoculates the medium. A rapidreaction is obtained because of the small volume of medium and the largeinoculum used. The identification of the unknown bacterium is achievedby determining a seven digit profile index number and consulting the APIprofile recognition system. However, there are strains of E. coli thatyield a low discrimination value with the API strips.

When this occurs, further identification with sugar test is required foraffirmation. Acid production from sugars such as D-Adonitol, Cellobiose,Lactose and D-Xylose are additional biochemical test for differentiationof Escherichia species and related species.

DNA Probes

The use of genetic probes in the detection of microorganisms is popularbecause they obviate the need for pure cultures, and are specific,sensitive, fast and reliable (Fred C. Tenover: DNA probes for infectiousdiseases. CRC Press, Inc. 1989). In DNA probe test, it is essential toknow something about the nucleotide sequence of the microorganisms underinvestigation.

Bacteria belonging to different families or strains can bedifferentiated on the basis of heterogeneity in genetic sequences. Oneapproach is the identification and use of specific toxin genes ofdisease causing strains to distinguish them from the normal flora.Another approach makes use of the conserved and polymorphic sites thatare found in bacterial 16S ribosomal RNA (rRNA) sequences not present inhuman 18 rRNA or human mitochondrial 12S rRNA. The combination of thepolymerase chain reaction technique for gene amplification, followed bysequencing of polymorphic regions and phylogenetic analysis of theresulting sequence information can also assist in strain identification.(Relman et al. The New Engl J of Medicine, 327: 293–301, 1992, Kui etal. FEMS Microbiology Letters 57:19–24, 1989. DeLong et al. Science 243:1360–1363, 1989).

The E. coli identification kit produced by gene-trak systems,Framingham, Mass., USA, uses DNA oligonucleotides that complement the16S rRNA. This assay uses hybridization techniques to detect E. coli,non-coli Escherichia fergusonii and Shigella species.

Another way of identifying bacteria specific DNA probes is by usingrandomly cloned chromosomal fragments. This involves the cloning ofrestriction enzyme cleaved genomic DNA of a bacteria, and selection ofspecific clones by determining their hybridization profiles byhybridization against its own species-sequences and otherspecies-sequences. Only clones that hybridize to sequences from the samespecies but the clones were derived from will be selected (Tenover FC:DNA Probes for Infectious Diseases. CRC Press 1989).

Gastrointestinal Infection

Colorectal cancer is one of the top three cancer killers in the world.Factors implicated in its etiology include inappropriate diet,environmental factors and lack of reliable diagnostic markers. Recently,greater understanding of the genetic predisposition to colon cancer hasbeen achieved through the identification of genes responsible for suchsusceptibility (Cowell J K, ed: In Molecular Genetics of Cancer. DunlopM G: Molecular genetics of colon cancer. 1995. 113–134). Despiteintensive research efforts, the mortality rate from colorectal cancerhas not declined dramatically over the last 40 years.

Markers associated with cancer initiation or progression are importantin patient care. Tumours diagnosed at an early stage can usually becured by surgical excision or polypectomy (surgical excision cures 90%of patients with adenoma or carcinomas that are confined to the mucosa).Patients with advanced disease have a poor prognosis as mortalityincreases to more than 90% after metastasis takes place.

The gastrointestinal tract is often exposed to a range ofmicroorganisms. When bacteria come into contact with a susceptible host,they can establish either a transient presence, colonize the individual,infect the individual or evolve with the host. The outcome can either beharmless, acute illness or a chronic condition that may lead to aserious outcome (Gibson G R and Macfarlane G T: Human Colonic Bacteria:Role in Nutrition, Physiology, and Pathology. CRC Press, Inc., 1995).

Bacteria have been associated with inflammatory bowel disease such asulcerative colitis and Crohn's disease (Giaffer et al. Gut 33:646–650,1992, Cartun et al. Mod Pathol 6:212–219, 1993; Liu et al.Gastroenterology 108:1396–404, 1995). In addition, patients withpan-colitis of long duration are at risk of developing colorectal cancer(Wanebo H J: In Colorectal Cancer. Lev R: Precursors of Colon Carcinoma1993; 158–163). Although frequently implicated, the role of bacteria incolon related disease remains ill-defined and controversial. Theidentification of bacteria in physical proximity to diseased tissue doesnot provide definitive proof of a causal relationship between abacterium and the diseased condition. This is especially so when thebacteria are commonly found surrounding the tissue (Swidsinski et al.Gastroenterology 115:281–286, 1998), as is the case in the colon, andthere is no additional information to differentiate between bacteria. Itis perhaps more convincing if the bacterium can be shown to bepositioned in-situ in the diseased tissue and when isolated andcharacterized found to possess properties that will substantiate itspresence within the tissue.

The bacterium Helicobacter pylori is an accepted Group 1 (definite)biological carcinogen for gastric cancer and causes of related gastricconditions such as duodenal ulcer, gastric ulcer and ulcercomplications. H. pylori attaches to and thrives on the gastric mucosaresulting in a chronic immunological response from the host. (Marshall,B. J. Gastroenterologist 1:241–247, 1993). It is not firmly establishedwhether H. pylori has invasive properties. However, pathogenic strainshave been identified that can cause epithelial cell damage and mucosalulceration on an intragastric administration to mice (Telford et al. JExp Med 179:1653–1658, 1994) The question remains whether H. pylori isthe only important factor in the development of gastric cancer becauseof its high infection/disease ratio. The current consensus is that theremay be other factors other than H. pylori infection that are alsoimportant in gastric cancer risk (National Institutes of HealthConsensus Development Panel on Helicobacter pylori in Peptic UlcerDisease 1994). A separate study put forward the theory that asynergistic interaction between a non-invasive bacteria and otherenteropathogens can facilitate invasion by the otherwise non-invasivebacteria (Geir Bukhowm and Georg Kapperud, Infection and Immunity55:2816–2821, 1987).

Numerous in-vivo and in-vitro studies have vividly shown thatmicroorganism carry transmissible tumorigenic genetic information.Mutagenesis in such instances is either by transposition orsite-specific recombination facilitated by conjugation, transformationand transduction. This information is constantly being exploitedscientifically in creating mutants (Sherratt D J (ed): Mobile geneticelements. Dale J W: Molecular genetics of bacteria. 2^(nd) Edition. JohnWiley and Sons Ltd. Oxford University Press 1995). In 1995, Couralin etal. showed that invasive strains of Shigella flexneri and E. coli cancarry out gene transfer that are stably inherited and expressed by themammalian cell progeny (Courralin et al., C. R. Acad. Sci. Paris318:1207–1212, 1995). Therefore, it is quite possible that thepersistent presence of bacterial genetic sequences in the nucleus ofmammalian cells can lead to genetic instability that may ultimately giverise to a tumour cell.

Bacterial invasion can stimulate similar a pattern of proteinphosphorylation to that induced by growth factor (e.g. EGF) and cellularproliferative responses may then be altered with consequences fordisease progression. (Galan et al. Nature 357:588–589, 1992). Inaddition, bacterial disruption of cell-cell interaction may affect cellproliferation patterns and differentiation (Epenetos A A and PignatelliM (ed): Cell Adhesion Molecules in Cancer and Inflammation; Pignatelliet al.: Adhesion molecules in neoplasia: An overview. Chapter 1:1–13.Harwood academic publishers 1995). Cytonecrotizing factors have beenidentified that can cause formation of large multinucleated cells andcells spreading in tissue cultures. (Denko et al. Experimental CellResearch 234:132–138, 1997; Lemichez et al., Molec Microbiol24:1061–1070, 1997; Machesky, L. M. and Hall, A, TICB 6:304–310, 1996).Accordingly, the persistence presence of bacteria can cause cellularchanges leading to cell disorientation, proliferation and changes incell morphology.

One cancer causing effect of bacteria is when Agrobacterium tumefaciens,a soil phytopathogen, genetically transforms plant cells by the transferof the tumour-inducing (Ti) plasmid to the plant genome where itsintegration and expression result in the crown gall phenotype. A crowngall is a tumorous proliferation of plant cells which are released fromnormal metabolic and reproductive controls (Hughes M A: Plant MolecularGenetics. Addison Wesley Longman Ltd. 1996).

People travelling across continents may suffer from traveler's diarrhoeaas the bacteria they are exposed to are not common in their county. Theassays/kits that are used for detecting microorganisms in theAsia-Pacific region are imported from other continents and theseimported assays/kits may not be as sensitive or as specific for thebacteria in the Asia-Pacific region.

Microorganisms transmitted by water and food usually grow in theintestinal tract of man and animals and leave the body in the faeces.Bacteria are known to possess gene sequences that make them toxigenic,hemorrhagic, invasive and adherent to tissues. Acute bacterial infectionis well documented but it is still not known that if bacteria that donot cause overt symptoms but persist and remain undetected in their hostcan cause diseases with time. Therefore, it is important that the assaysthat are available are sensitive and specific for a wide range ofpathogens.

The E. coli genetic sequence is published. (Blattner et al. Science277:1453–1474, 1997). Some of its genetic sequence has homology to otherbacteria (Janda J M and Abbott S L: The Enterobacteria. Lippincott-RavenPress 1998). The inventor, in accordance with the present invention, hasidentified E. coli DNA sequences which are unique to the Escherichaefamily and furthermore has shown that biochemical and cultural testspresently available are not adequate for detecting this family ofbacteria. The present polynucleotide sequence in the genome of strainsof the Escherichieae genus (Escherichia and Shigella), have proven to bemore informative than the agar plates EMB, MacConkey and MUG. They canbe used to detect E. coli that is either EMB, MacConkey or MUG negative.The sequences are also found in 0157:H7 and 029:NM strains of E. coli.Therefore, the present molecular markers provide improved tools for thedetection and characterization of E. coli.

In addition, the invention permits the use of the sequence(s) to studythe outcome of tissue infection in-situ. The present gene sequences aremore specific than the gene-trak sequence (gene-trak systems) and thesequences can be amplified many-fold to increase their detection limit.This makes the present invention useful for studying the role ofmicroorganism in gastrointestinal and other disease conditions. Thepresence of the polynucleotide sequence in cells can be located by theuse of the polymerase-chain-reaction amplification technique in-situfollowed by hybridization to the in-situ amplified signals with sequencespecific DNA probe.

The identification of these specific polynucleotide sequence(s) that canbe used to detect for the presence of strains of E. coli and Shigellaand related microorganisms in food, water, fecal specimens, tissues,secretions and other biological, environmental and/or laboratory samplesis important for health reasons as it enables one to check on thequality of food and water hygiene and monitor transmission of themicroorganism. Sensitive detection techniques and methods for assessingthe role of bacteria in clinical conditions will ultimately help in thecontrol of harmful microorganisms.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

One aspect of the present invention provides a new use for the whole ofFormula I and sequences within Formula I, as markers for species ofbacteria within the Escherichieae family specifically Escherichia coliand Shigella species or related microorganisms.

Another aspect of the present invention relates to the use of thepolynucleotide sequence of formula I to generate gene probes of smallersize which singularly or in combination have specificity for E. colistrains or related microorganisms but not necessarily specific for allthe Shigella species.

A further aspect of the present invention provides Formula I and thesmaller gene sequences within it as a means to detect the presence, inliquids, semisolids and solids combinations thereof or in aerosols orgases, of species of bacteria within the Escherichieae familyspecifically E. coli and some or all of the Shigella species so thathigh standard of sanitation can be achieved.

Yet another aspect of the present invention provides the aforementionedsequence(s) as a means to detect infection in a sample and/or acombination of samples by members of the Escherichieae family asaforementioned. Samples are defined in this invention as tissues orcells or explants of either human, animals or plant origin, such givenexamples being tissue/cells found in the colon, stomach, and other partsof the human or animal anatomy as well as in food, industrial and/orenvironmental samples.

A further aspect of the present invention provides a method for testingand identifying the various genes within formula I as new means todetect for changes in DNA content in cells infected or previouslyinfected with the aforesaid Escherichieae family of bacteria (E. coliand Shigella species or related microorganisms). A cell is defined inthis invention as a cell found in the animal and plant kingdom. Changesin DNA content in a cell in accordance with this invention includes DNAsequences found in the cell which differs by one or more nucleotidesubstitutions, additions and/or deletions of existing DNA or by theintroduction of a heterologous DNA.

Yet another aspect of the present invention provides the aforementionedFormula I within which polynucleotide sequences are a marker for use inrecognizing early cellular DNA changes associated with any one or moremembers of the Escherichieae family (E. coli and Shigella species andrelated microorganisms) in the colonic epithelium before the histologycriteria for such cellular changes are detectable. Early changes aredefined in this invention by the presence of at least bacterial DNAsequences that are present in high, low copy numbers or present assingle copies per haploid genome in a normal population.

Still yet another aspect of the present invention provides theaforementioned Formula I within which polynucleotide sequences are amarker for use in recognizing pre-malignant changes associated with anyone or more members of the Escherichieae family (E. coli and Shigellaspecies and related microorganisms) in the colonic epithelium as definedby histology criteria for such pre-malignant tissue. Pre-malignantchanges are defined in this invention by the presence of at leastbacterial DNA sequences that are present in high or low copy numbers orpresent as single copies per haploid genome in a normal population andare supported by histology criteria.

Even still another aspect of the present invention provides theaforementioned Formula I within which polynucleotide sequences can be amarker for use in recognizing malignant changes associated with any oneor more members of the Escherichieae family (E. coli and Shigellaspecies and related microorganism) in the colonic epithelium andmalignant colonic tumours residing in other tissues. Malignant changesare defined by histology criteria. Malignant changes associated with anyone member of the Escherichieae family are defined in this invention bythe presence of at least bacterial DNA sequences that are present inhigh or low copy numbers or present as single copies per haploid genomein a normal population.

Another, aspect of the present invention provides the aforementionedformula I within which polynucleotide sequences can be used as a markerfor detecting pre-malignant changes associated with any one or moremembers of the Escherichieae family in the gastric mucosa as defined byhistology criteria for such pre-malignant tissues. Pre-malignant changesare defined in this invention by the presence of at least bacteria DNAsequences that are present in high or low copy numbers or present assingle copies per haploid genome in a normal population and itshistology criteria for the tissue defined.

Yet another aspect of the present invention provides the aforementionedFormula I sequence within which polynucleotide sequences can be used asa marker to recognize malignant changes associated with any one or moremembers of the Escherichieae family in the malignant gastric tumours andmalignant gastric tumours residing in other tissues. Malignant changesare defined inter alia by histology criteria. Malignant changesassociated with the Escherichieae family in this invention is defined bythe present of at least DNA sequences that are present in high or lowcopy numbers or present as single copies per haploid genome in a normalpopulation.

Still another aspect of the present invention provides theaforementioned Formula I within which sequences can be used as markersto recognize patients that are found harboring any one or more member ofthe Escherichieae family relative to normal patients not haboring thesame and are thus identified as marker of infection of said family thatare important in patient care.

Even still another aspect of the present invention provides theaforementioned Formula I sequence within which polynucleotide sequencescan be used as markers being found in colorectal cancer patientsrelative to normal patients and thus identified as a marker of malignantdisease that is important in patient care.

Even yet another aspect of the present invention provides theaforementioned marker that is found in gastric cancer patients relativeto normal patients and is thus identified as a marker of malignantdisease that is important in patient care.

Another aspect of the present invention provides a marker for cellularinstability and therefore a marker for predisposition to cellularcarcinogenesis. Cellular instability may occur as a forerunner tocellular carcinogenesis or other condition and is characterized hereinby changes in DNA content comprising one or more nucleotidesubstitutions, additions and/or deletions of existing DNA or by thepresence of heterologous DNA.

Another aspect of the present invention provides a method of testing andselecting sequences in E. coli and Shigella species and relatedmicroorganisms as markers for use to detect changes in DNA content incells in order to recognize cellular instability and, therefore,predisposition to cellular carcinogenesis, predisposition to colon andgastric cancer and as markers for use in recognizing benign,pre-malignant and malignant gastrointestinal tissues as optionallydefined by histology criteria.

In accordance with the present invention, it is shown that Formula Icomprises polynucleotide DNA sequence marker(s) for the Escherichieaefamily specifically E. coli species and Shigella species or relatedmicroorganisms. This Formula I and the various genes and sequence itcontains allow the differentiation of the aforementioned members of theEscherichieae family from other bacteria families. In addition, thepresence of such bacteria as indicated by the presence of the DNAsequences allows study of sanitation and health related matters such asinfection, predisposition to cancer, cancer and cell instability.

Reference to “related microorganisms” includes microorganisms which arerelated at the immunological, biochemical, disease-causing,physiological or genetic levels. A derivative or mutant form of E. colior Shigella species is an example of a related microorganism.

The present invention furthermore provides a method of testing andselecting other sequences in E. coli and Shigella species and relatedmicroorganisms as markers to test for their presence in cells withabnormal cell growth or physiology associated with cancer or apredisposition to the development of cancer.

Yet still another aspect of the present invention relates to a new usefor the various polynucleotide sequences within Formula I as molecularprobes in the determination of whether samples contain members of theEscherichieae family such as E. coli and Shigella species or relatedmicroorganisms.

In another aspect, the present invention provides methods forenhancement in the specificity and sensitivity of detecting thepresence, among other bacteria of E. coli species and some of theShigella species with some of the aforementioned sequences.

The presence of the polynucleotides sequences in food and water isevidence that they are contaminated with members of Escherichieae familysuch as E. coli species and probably some or all of the Shigellaspecies. Thus the present molecular probes provide an alternative tomicrobiological and biochemical assays which are less specific,sensitive, reliable, often required for pure cultures, and are more timeconsuming.

A further related aspect of the present invention provides a new use forthe sequences within the Formula I for determining whether tissuesamples contain the DNA markers that originate from members of the E.coli and Shigella species. Both species within this family are known tohave invasive, adherent and toxigenic properties. This aspect relates tothe new use of polynucleotide sequence(s) within Formula I as marker(s)for detecting infection by identifying samples such as, for example,colonic and gastric mucosa tissues that contain them. The presence ofthe polynucleotide sequences in tissues is evident that the tissues areinfected by members of the E. coli and Shigella species.

A further aspect of the instant invention relates to a new use for thesequences within the Formula I for determining which cell type withintissues samples contain marker DNA sequences that originate from membersof the Escherichieae family such as E. coli and the Shigella species andrelated microorganisms.

A further aspect of the instant invention relates to a new use for theaforementioned polynucleotide sequence(s) within Formula I as a maker(s)for detecting changes in cellular DNA composition in, for example,colonic and gastric mucosa cells before histology criteria for changesare detectable. The presence of the polynucleotide sequences in cells oftissues is evidence that the cells are infected by members of the E.coli and Shigella species. Changes in cellular DNA composition aredefined in this particular aspect of the invention by the presence of atleast bacteria DNA sequences that are present in high or low copynumbers or present as single copies per haploid genome in a normalpopulation. The polynucleotide sequence of the present invention areonly found in Escherichieae family and, therefore, their presence inother species such as in eukaryotic cells, for an example, is a sign ofan abnormal event. Accordingly, the present invention provides one ormore molecular marker for screening patients to identify those who areat risk of having gastrointestinal tumours (benign, pre-malignant, ormalignant).

A further additional aspect of the present invention relates to a newuse for the sequences within the Formula I for determining whetherpre-malignant tumours as defined by histology criteria containaforementioned polynucleotides sequences that originate from members ofthe E. coli and Shigella species or related microorganisms. This aspectrelates to the new use of the marker for detecting the presence of anyone or more member of the Escherichieae family in the pre-malignanttumours such as colonic and gastric tissues as defined by histologycriteria. The presence of the polynucleotide sequence in the cells ofpre-malignant colonic and gastric tumours is evidence that the cells areinfected by or contain DNA sequences of members of the E. coli andShigella species. These pre-malignant tumours contain the presence of atleast bacteria DNA sequences that are present in high or low copynumbers or present as single copies per haploid genome in a normalpopulation. The polynucleotide sequence of the present invention areonly found in Escherichieae family and, therefore, their presence inother species such as in eukaryotic cells, for an example, is a sign ofan abnormal event. Accordingly, the present invention provides one ormore molecular markers for screening patients to identify those at riskof having gastrointestinal tumours (benign, pre-malignant, malignant).

The present invention also relates to a new use for the aforementionedpolynucleotide sequence(s) as a marker for determining whether malignantchanges in the colonic and gastric mucosa contain marker sequences thatoriginate from members of the E. coli and Shigella species. Malignantchanges are defined by conventional histological criteria. This aspectof the invention relates to the new use of the marker for detecting theDNA presence of any one or more member of the Escherichieae family inthe malignant tumours. The presence of the polynucleotide sequence inthe cells of these malignant tumours is evidence that the cells areinfected by or contain DNA sequences of members of the E. coli and/orShigella species. These malignant tumours contain the presence of atleast bacteria DNA sequences that are present in high, low copy numbersor present as single copies per haploid genome in a normal population.The polynucleotide sequence of the present invention are only found inEscherichieae family and, therefore, their presence in other speciessuch as in eukaryotic cells, for an example, is a sign of an abnormalevent. Accordingly, the present invention provides one or more molecularmarkers for screening patients having gastrointestinal tumours (benign,pre-malignant, malignant).

The instant invention provides in a related embodiment a new use for theaforementioned polynucleotide DNA sequence(s) as markers for determiningthe presence of any member of the E. coli and Shigella species inmetastatic cells of colonic or of gastric tumour origin residing inother tissues. This aspect of the invention relates to the new use ofthe marker sequence for detecting the DNA presence of any one or moremember of the E. coli and/or Shigella species in the metastatic cells.The presence of the polynucleotide sequence in the cells of thesemalignant tumours is evidence that the cells are infected by or containDNA sequences of members of the E. coli and/or Shigella species. Thesemetastatic cells contain the presence of at least bacteria DNA sequencesthat are present m high or low copy numbers or present as single copiesper haploid genome in a normal population. The polynucleotide sequenceof the present invention are only found in Escherichieae family and,therefore, their presence in other species such as in eukaryotic cells,for an example, is a sign of an abnormal event. Accordingly, the presentinvention provides one or more molecular markers for screening patientshaving gastrointestinal metastatic cells.

The present invention furthermore relates in a different aspect to a newuse for the formula I sequence that is found in E. coli and Shigellaspecies or in related microorganisms, as a marker for determining cellsthat possess it. The invention relates to the new use of the markersequence for detecting the DNA presence of any one or more member of theE. coli and/or Shigella species in the cells. This gene sequence is onlyfound in Escherichieae family and therefore its presence in high, lowcopy numbers or as single copies per haploid genome in a normalpopulation in eukaryotic cells, for an example, is a sign of an abnormalevent that may lead to genetic instability of cell that possess it. Itprovides as a molecular marker for risk of and genetic instability andtherefore tumourigenesis.

Even more particularly the present invention, in one aspect, provides amethod for detecting the presence of E. coli or Shigella species orrelated microorganisms in a sample, said method comprising subjecting anucleic acid molecule preparation from said sample to genetic analysisusing one or more E. coli- or Shigella species'-specific nucleotidesequences obtainable from one or more nucleotide sequences of Formula 1and/or Table 1 wherein the ability for said E. coli- or Shigellaspecies'-specific nucleotide sequences to hybridize to complementarynucleotide sequences in the nucleic acid preparation is indicative ofthe presence of E. coli, Shigella species or related microorganisms.

In a further aspect of the present invention there is provided a methodfor detecting the presence of E. coli and/or Shigella species or relatedmicroorganisms in a sample as hereinbefore described wherein thenucleotide sequences of Formula I comprises from nucleotide position 246of GenBank Accession No. AE000201 to nucleotide position 6693 of GenBankAccession No. AE000203 including the nucleotide sequence of GenBankAccession No. AE000202.

Still a further aspect of the present invention provides a method fordetecting the presence of E. coli or Shigella species or relatedmicroorganisms in a sample as hereinbefore described wherein the E.coli- and/or Shigella species'-specific nucleotide sequences comprisesat least 8 nucleotides in length.

A related aspect of the present invention discloses a method fordetecting the presence of E. coli or Shigella species or relatedmicroorganisms in a sample as hereinbefore described whereinhybridization of E. coli- and/or Shigella species'-specific nucleotidesequences to the nucleic acid preparation is detected by the presence ofamplified nucleic acid products.

A further related aspect of the present invention provides a method fordetecting the presence of E. coli, Shigella species or relatedmicroorganisms in a sample wherein hybridization of E. coli- and/orShigella species'-specific nucleotide sequences to the nucleic acidpreparation or the presence of amplified nucleic acid products isdetected by a reporter molecule giving an identifiable signal.

Still yet a further related aspect of the present invention provides amethod for detecting the presence of E. coli, Shigella species orrelated microorganisms in a the sample wherein the sample comprisesfood, water, semi-solids or semi-liquid material, mammalian tissue,tissue extract or cells of tissue or normal tissue or tissue predisposedto cancer growth or malignancy or cellular instability.

In a particularly preferred aspect of the present the mammalian tissueis associated with colon, stomach or colorectal tissue.

A related aspect of the present invention provides a method foridentifying nucleotide sequences, or their expressed products, capableof inducing or otherwise facilitating abnormal cell growth or abnormalphysiology, said method comprising introducing a nucleotide sequencecomprising E. coli- and/or Shigella species'-specific nucleotidesequences from the nucleotide sequences in Formula I into cells andobserving morphological and/or physiological changes to said cellscompared to control cells without said introduced nucleotide sequenceswherein the presence of abnormal morphology and/or physiology in a cellis indicative of a nucleotide sequence from Formula I, or a polypeptideexpressed therefrom, which is capable of inducing or facilitatingabnormal cell growth or physiology.

In a further preferred aspect of the instant invention the abnormal cellgrowth or physiology is associated with cancer or a predisposition tothe development of cancer or cellular instability.

A further aspect of the present invention provides a molecular probecomprising at least 8 nucleotides obtainable from the nucleotidesequences of Formula I wherein said molecular probe is capable ofspecifically hybridizing to E. coli- and/or Shigella species'-derivednucleic acid molecules.

Yet more particularly the present invention encompasses a use of anucleotide sequence obtainable from the nucleotide sequence of Formula Iin the manufacture of a molecular probe for the identification of E.coli and/or Shigella species and/or for the identification of a cellularinstability or a cancer or tumor or a predisposition to development ofsame.

Still even yet more especially the present invention provides a methodfor testing and selecting other sequences in E. coli, Shigella speciesor related microorganisms in a sample, said method comprising subjectinga nucleic acid molecule preparation from said sample to genetic analysisusing one or more E. coli or Shigella species'-specific nucleotidesequences obtainable from one or more nucleotide sequences of Formula 1and/or Table 1 wherein the ability for said E. coli- or Shigellaspecies'-specific nucleotide sequences to hybridize to complementarynucleotide sequences in the nucleic acid preparation is indicative of anE. coli or Shigella species'-specific nucleotide sequence.

Another aspect of the instant invention provides a molecular probe of atleast 8 nucleotides, identified by the methods as herein describedwherein said probe comprises a sequence of nucleotides from Formula Iand wherein said molecular probe is capable of specifically hybridizingto E. coli and/or Shigella species'-derived nucleic acids.

The invention still yet provides the use of a nucleotide sequenceidentified by the methods herein disclosed in the manufacture of amolecular probe for the identification of E. coli, Shigella speciesand/or for the identification of a cellular instability or a cancer ortumour or a predisposition to development of same.

A related aspect of the instant invention discloses a use of anucleotide sequence specific to E. coli and/or Shigella species and/orrelated microorganism in the manufacture of a molecular probe for theidentification of one or more gastrointestinal cancers or tumours or apredisposition to the development of same.

A final preferred aspect of the instant invention provides a molecularprobe comprising a nucleotide sequence specific to E. coli and/orShigella species and/or related micro-organism for the identification ofone or more gastrointestinal cancers or tumours or a predisposition tosame.

Other aspects, features and advantages of the present invention willbecome apparent from the detailed description that follows, or may belearned by practice of the invention.

For the sale of brevity, reference to specific microorganisms such asEscherichia coli (E. coli) or Shigella species includes reference torelated microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures serve to further explain the principles of theinstant invention. It is to be understood, however, that the figures aredesigned for purposes of illustration only, and not as a definition ofthe limits of the invention for which reference should be made to theclaims appearing at the end of the description.

FIGS. 1 a–c. Schematic diagram of the various locations of the genestested that is within the polynucleotide sequence of formula I. Theformula I sequence extends from nucleotide position (nt) 246 of GenBankaccession #AE000201, including sequence of GenBank accession #AE000202to nucleotide position 6693 of GenBank accession #AE000203.

FIG. 2. Autoradiograph result of probe A hybridizing to a panel ofbacteria DNA as listed in Table 2a grid C. Probe A consists of fragments1,2,3 and 4 as depicted in FIG. 1, a–c. Each fragment is generated byprimer directed PCR carried out on K12 E. coli DNA and subsequentlycombined for ³²P labeling and hybridization. The gene sequence spansbetween nucleotide position 1163 of AE000201 through AE000202 to 503 ofAE000203. The primer pairs used are: ECM-1163, torT-5750 (fragment 1,AE000201); torT-5129 AE000201, CD-1351 AE000202 (fragment 2); CD-415,ycdG 7359 (fragment 3, AE000202); ycdG-6073 AE000202, New2-503 AE000203(fragment 4). Five hundred nanogram DNA is loaded per dot. Posthybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 3. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 246 to 850 of AE000201. ECM-246 and ECM-850are the primers used to generate the gene probe by PCR amplification ofK12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM NApyrophosphate at 65° C.

FIG. 4. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 1163 to 1958 of AE000201. ECM-1163 andECM-1958 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 5. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 7218 to 7761 of AE000201. Primers tor C-7218and tor C-7761 are used to generate the gene probe by PCR amplificationof K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 6. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 8332 to 8891 of AE000201. Primers tor A-8332and tor A-8891 are used to generate the gene probe by PCR amplificationof K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 7. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 10574 to 11160 of AE000201. Primers torD-10574 and tor D-11160 are used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 0.1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 8. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 415 to 1351 of AE000202. CD-415 and CD-1351are the primers used to generate the gene probe by PCR amplification ofK12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 0.1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 9. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 3151 to 4359 of AE000202. Primers agp-3151and agp4359 are used to generate the gene probe by PCR amplification ofK12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 10. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid C. The gene sequence spansbetween nucleotide position 4807 to 5235 of AE000202. Wrb-4807 andWrb-5235 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 11. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 6073 to 7359 of AE000202. Primers ycdG-6073and ycdG-7359 are used to generate the gene probe by PCR amplificationof K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 0.1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 12. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid A. The gene sequence spansbetween nucleotide position 7223 to 7794 of AE000202. 81B-7223 and81B-7794 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 13. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 7278 to 7773 of AE000202. 81B-7278 and81B-7754 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 14. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid C. The gene sequence spansbetween nucleotide position 7419 to 7985 of AE000202. OH-7419 andOH-7985 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 15. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 7562 to 7794 of AE000202. OH-7562 and81B-7794 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 16. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid C. The gene sequence spansbetween nucleotide position 8160 to 9704 of AE000202. New1-8160 andNew1-9704 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 MM Na pyrophosphate at 65° C.

FIG. 17. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 9731 to 11375 of AE000202. New2-9731 andB-11375 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 5×SSC, 0.05% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 18. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid C. The gene sequence spansbetween nucleotide position 9731 of AE000202 to 503 of AE000203. New2-9731 and New2-503 are the primers used to generate the gene probe byPCR amplification of K12 E. coli genomic DNA. Five hundred nanogram DNAis loaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 19. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid B. The gene sequence spansbetween nucleotide position 5944 to 6693 of AE000203. Primers putP-5944and putP-6693 are used to generate the gene probe by PCR amplificationof K12 E. coli genomic DNA. Five hundred nanogram DNA is loaded per dot.Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 MM Napyrophosphate at 65° C.

FIG. 20. Autoradiograph result of radiolabeled probe A hybridized toEnterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2agrid E. Post hybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mMNa pyrophosphate at 65° C.

FIG. 21. Autoradiograph result of radiolabeled gene probe hybridized toEnterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2agrid E. The gene sequence spans between nucleotide position 7562 to 7794of AE000202. OH-7562 and 81B-7794 are the primers used to generate thegene probe by PCR amplification of K12 E. coli genomic DNA. Posthybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 22. Autoradiograph result of radiolabeled gene probe hybridized toEnterobacter cloacae and K12 E. coli genomic DNA as depicted in Table 2agrid E. The gene sequence spans between nucleotide position 7223 to 7794of AE000202. 81B-7223 and 81B-7794 are the primers used to generate thegene probe by PCR amplification of K12 E. coli genomic DNA. Posthybridization wash condition is 1×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate at 65° C.

FIG. 23. Autoradiograph result of radiolabeled gene probe hybridized tobacteria DNA as listed in Table 2a grid D. The gene sequence spansbetween nucleotide position 7278 to 7773 of AE000202. 81B-7278 and81B-7754 are the primers used to generate the gene probe by PCRamplification of K12 E. coli genomic DNA. Five hundred nanogram DNA isloaded per dot. Post hybridization wash condition is 1×SSC, 0.1% w/vSDS, 20 mM Na pyrophosphate at 65° C.

FIG. 24 Autoradiograph result of ³²P radiolabeled H. pylori ribosomalgene probe hybridized to bacteria DNA as listed in Table 2a grid A.Primer pairs indicated are used for PCR amplification of H. pylorigenomic DNA to generate the required gene segment. Five hundred nanogramof genomic DNA is loaded per dot. Post hybridization wash condition is0.1×SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65° C.

FIG. 25. Autoradiograph result of ³²P radiolabeled H. pylori ribosomalgene probe hybridized to bacteria DNA as listed in Table 2a grid D.Primer pairs indicated are used for PCR amplification of H. pylorigenomic DNA to generate the required gene segment. Five hundred nanogramof genomic DNA is loaded per dot. Post hybridization wash condition is0.1×SSC, 0.1% w/v SDS, 20 mM Na pyrophosphate at 65° C.

FIG. 26. Autoradiograph result of in vitro simulated PCRISH. H. pylori³²P radiolabeled ribosomal gene probe is hybridized to productsgenerated from its primer directed PCR amplification of H. pylori and E.coli genomic DNA and total DNA of H. pylori and E. coli isolatesobtained from patients' fecal specimens. The post hybridization washcondition is 5×SSC, 0.05% w/v SDS, 20 mM Na pyrophosphate at 65° C. SeeTable 2a grid F.

Table 1. Oligonucleotide Primers.

Table 2a. Grids A,B,C show the different types of bacteria genomic DNAloaded onto corresponding nylon plus membrane and hybridized to randomprimed ³²P-radiolabeled gene probes. Columns W to Y for all three gridshave the same panel of bacteria DNA. Column Z as indicated, has only afew bacteria DNA that is common among them. DNA of E. coli isolatesobtained from patient fecal specimens are: 219/1, 196/1, 196/28, 197/5,218/40, 142/31, 179/36, and 117/3B. Patient's Shigella sonnei isolate is219/1. 078:H11 and 0157:H7 are commercial E. coli strains. TG2 is a giftfrom Gibson T J. Placental DNA is from commercial source (Sigma, UK).SssDNA is sonicated denatured salmon sperm DNA (Sigma, UK). Thesemembranes exist in replicates and are hybridized to differentradio-labeled gene probes. 500 ng DNA is loaded per dot.

Table 2a. Grid D correspond to nylon plus membrane that contain DNA fromE. coli and gram positive bacteria isolated from patients' fecalspecimen. Unless otherwise stated, all are E. coli DNA. Placental DNA,K12 and 0157:H7 are from commercial source. 114/3 g is Streptococcusgroup D DNA, 115/TA is Streptococcus group G DNA, 116/TC is Aeromonasssobria DNA, 116/TD is Streptococcus viridans DNA, 117/2D isStreptococcus group D DNA, 154/9 is unidentified gram positive bacteriaDNA and HP is Helicobacter pylori DNA. 500 ng DNA is loaded per dot.

Table 2a. Grid E correspond to nylon plus membranes that contain induplicate a range of different amount of Enterobacter cloacae and K12 E.coli DNA. The membranes are then hybridized to different³²P-radiolabeled gene probes to determine the level of crossover betweenthe 2 different species of bacteria.

Table 2a. Grid F correspond to nylon plus membrane that containsdifferent amount of E. coli DNA, H. pylori DNA and H. pylori primerdirected PCR product of E. coli and H. pylori DNA. This is hybridized to³²P radiolabeled H. pylori ribosomal gene probe (HP).

Table 2b. Bacteria source.

Table 3. Result of in-vitro simulation of PCRISH. The primer directedPCR amplification of bacteria DNA has resulted in amplification ofproduct(s). This is indicated by the appearance of band(s) upon gelresolution of a given aliquot of a post PCR mixture (not shown). PostPCR mixture having multiple bands is denoted as positive if any one DNAband migrates at the expected molecular weight of the intended targetproduct. Hybridization with corresponding radiolabeled gene probe hasnot picked up any non-specific bands other than the single band of theintended target.

FIGS. 27–34: Detection of bacteria DNA in biopsies and surgicalspecimens obtained from the colon bypolymerase-chain-reaction-in-situ-hybridization technique (PCRISH). Forthe detection of 81B gene sequence (marker for E. coli/Shigella speciesexcept Shigella boydii) PCR in-situ amplification is carried out withouter primers 81B-7223 and 81-7794. The PCR digoxigenin labeled 81B geneprobe is made with inner primers 81B-7278 and 81B-7754. For detection ofH. pylori ribosomal gene sequence, PCR in situ amplification is carriedout with the outer primers HP-178 and HP-775. The PCR digoxigeninlabeled H. pylori Probe is made with inner primers HP-228 and HP-513.Positive signals are denoted by the dark spots and an example ishighlighted by the arrow. Tissue condition is defined by histologiccriteria.

FIG. 27: Hyperplastic polyp with no evidence of malignancy tested with81B probe.

FIG. 28: Adenomatous polyp, tubulovillous type tested with 81B probe.

FIG. 29: Well differentiated adenocarcinoma tested with 81B probe.

FIG. 30: Liver tissue: metastatic, poorly differentiated adenocarcinomawith primary in the gastrointestinal/pancreatico-biliary tract testedwith 81B probe.

FIG. 31: Normal mucosa next to tumour tested with 81B probe.

FIG. 32: Normal mucosa tested with 81B probe.

FIG. 33: Colonic mucosa diagnosed with proctitis tested with 81B probe.

FIG. 34: Liver specimen: metastatic, poorly differentiatedadenocarcinoma with primary in the gastrointestinal/pancreatico-biliarytract. Specimen tested negative with H. pylori ribosomal gene probe.

FIGS. 35–44: PCRISH detection of 81B DNA (marker for E. coli/Shigellaexcept Shigella boydii) and H. pylori DNA in biopsies and surgicalspecimens obtained from the stomach. H. pylori Probe has not been testedagainst closely related Helicobacter species, and therefore does notclaim to detect only H. pylori. Positive signals are denoted by the darkspots and an example is highlighted by the arrow. PCR in-situamplification is carried out with outer primers 81B-7223 and 81B-7794for the detection of the 81B gene and HP-178 and HP-775 for thedetection of H. pylori gene. The PCR digoxigenin labeled 81B gene probeis made with inner primers 81B-7278 and 81B-7754, while HP-228 andHP-513 Primers are used for the H. pylori gene probe. Tissue conditionis defined by histologic criteria.

FIG. 35: Adenocarcinoma of stomach tested with 81B probe.

FIG. 36: Normal gastric mucosa adjacent to gastric tumour tested with81B probe.

FIG. 37: Liver specimen: metastatic, poorly differentiatedadenocarcinoma from gastric cancer, tested with 81B probe.

FIG. 38: H. pylori negative normal gastric mucosa tested with 81B probe.

FIG. 39: H. pylori negative normal gastric mucosa tested with 81B probe.Specimen is the same as the one shown in FIG. 38 but showing a differentarea.

FIG. 40: Normal gastric mucosa adjacent to gastric tumour tested with H.pylori Probe.

FIG. 41: Adenocarcinoma of stomach tested with H. Pylori probe.

FIG. 42: Liver specimen: metastatic, poorly differentiatedadenocarcinoma from gastric cancer tested with H. pylori Probe.

FIG. 43: Active chronic gastritis in the presence of H. pylori andtested with H. pylori probe.

FIG. 44: Active chronic gastritis in the presence of H. pylori, testedwith 81B probe.

FIG. 45: GenBank Accession Nos. AE000201, AE000202 and AE000203comprising respectively sections 91, 92 and 93 of 400 of the completegenome sequences of Escherichia coli K-12.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein identifies inter alia a new use for Formula I andnucleotide sequences that are part of Formula I (FIG. 1, a–c) asmolecular markers. The Formula I sequence extends from nucleotideposition (nt) 246 of GenBank accession #AE000201, including sequence ofGenBank accession #AE000202 to nucleotide position 6693 of GenBankaccession #AE000203.

The various DNA fragments for use as hybridization probes can begenerated by polymerase chain reaction (PCR) using the primer sequencestabulated in Table 1. In this invention the various DNA fragments areshown to have a new use for identifying bacteria in the Escherichieaefamily such as E. coli and Shigella species. Each DNA fragment generatedis specific for E. coli species and sorne Shigella species as some ofthe gene sequences show different specificity towards Shigella boydiiand Shigella Flexneri (Table 2a, FIG. 2–19).

The DNA fragments highlighted in this invention as examples, andincluding Formula 1, can be use in detecting the aforementioned bacteriapresence in liquids, solids, semi-solids to identify the level ofsanitation, in monitoring the level/depth of infection, in studying theassociation of the presence of sequences within Formula I ingastrointestinal conditions or other clinical conditions where theirpresence/aforementioned bacteria is sought. The invention findsparticular utility in monitoring for the presence of metastatic gastricand/or colon tumour cells. In addition, it can be used to study theassociation of such aforementioned sequences with gastrointestinalcancer risk. In addition, it can be used for studying the stability ofgenome sequence in cells that contains sequences of Formula I whether inparts, complete or in association with sequence upstream or downstreamof Formula I as can be obtained in the published sequence of E. colistrains (example of source: GenBank database). Furthermore, theinvention shows that when the sequences of Formula I are found insidecells of tissues, it is useful to identify specimens where the pathogencan be isolated and identified by probing with gene sequences forpathogenic properties, especially when the pathogen does not cause overtsymptoms.

Accordingly, one aspect of present invention provides a method fordetecting the presence of E. coli or Shigella species or relatedmicroorganisms in a sample, said method comprising subjecting a nucleicacid molecule preparation from said sample to genetic analysis using oneor more E. coli- or Shigella species'-specific nucleotide sequencesobtainable from one or more nucleotide sequences of Formula 1 and/orTable 1 wherein the ability for said E. coli- or Shigellaspecies'-specific nucleotide sequences to hybridize to complementarynucleotide sequences in the nucleic acid preparation is indicative ofthe presence of E. coli, Shigella species or related microorganisms.

In a particularly preferred embodiment Formula I comprises fromnucleotide position 246 of GenBank Accession No. AE000201 to nucleotideposition 6693 of GenBank Accession No. AE000203 including the nucleotidesequence of GenBank Accession No. AE000202.

Specificity of DNA Probe.

The invention herein describes Formula I and the various genes itencodes, which characteristically permit identification of members ofthe Escherichieae family especially E. coli species and Shigellaspecies.

The various DNA gene probes are generated by primer directed PCRamplification carried out on E. coli genomic DNA. The choice of primersequence is assisted with the use of the software programme, PCR PLAN(PC/Gene system, Intelligenetics, Inc. USA) as applied on the publishedsequence of E. coli obtained from GenBank database accession #AE000201,#AE000202, and #AE000203. The suitability of a gene probe will depend onits specificity. The specificity of the primer sequence and gene probewill have to be determined empirically in a step-wise systematic fashionalong the Formula I sequence or by use of software programmes. The genefragment generated by primer directed PCR or the primer sequence shouldpreferably be without degeneracy when tested on the panel of bacteria ofrelated species. For the gene fragment, any variation observed isfurther characterized so that the variation is consistent with aparticular strain or species (e.g. E. coli versus Shigella) but yethaving homology to the test probe. Each gene probe fragment ispreferably purified after amplification to ensure specificity.

There are many ways of obtaining such bacteria-specific fragments: thiscan be achieved, for example, by cloning the bacteria DNA, either inparts or complete, and screening with oligonucleotide or genomic probesof E. coli. The species or strain specificity of the cloned probes isdetermined.

The dot blot format (96 well dot-blot manifold, Bio-Rad, USA) is usedhere for creating a panel of bacteria genomic DNA to test for thespecificity of the DNA probes (Table 2a). Reference strains are obtainedfrom American Type Culture Collection (Table 2b. ATCC, Rockville, Md.,USA). A representative bacteria from each member of theenterobacteriaceae family is chosen for the panel on which the probesare to be tested against. Gram-negative and gram-positive microorganismsobtained from subjects/patients are used as test samples (Table 2a, gridD). The reference strain and test strains are streaked for isolation,colony purified, and verified by analytical profile index test strips(API, bioMerieux, USA) and if required additional sugar test (acidproduction test from D-Mannitol, Cellobiose, Lactose and D-Xylose). Thepanel of bacteria chosen include organisms that are likely to be foundin the natural flora of food and fecal specimens or have known orsuggested physiologic or genetic relatedness to E. coli and Shigellaspecies.

The nucleotide sequence of the present invention may be a non-ribosomalsequence of E. coli, Shigella species or a related species.

The DNA probes are ³²P-labeled by random prime method (AmershamPharmacia Biotech Inc, USA ) as per supplier's instruction but withmodifications. The probe specificity is refined carefully by controllingthe hybridization condition and the post hybridization wash condition.It will be noticed herein, that for optimal specificity, the posthybridization salt wash condition ranges between 5×SSC and 0.1×SSC forthe various probes. Unless specified, the wash temperature for themembranes is at 65° C.

Probe A is made up of a combination of fragments (1,2,3 and 4 of FIG. 1,a–c) and covers a large part of the Formula I sequence. It will benoticed that probe A and some of the gene probes within Formula I (Table2a, FIGS. 2–19) are specific for the E. coli species and some Shigellaspecies, implying that there are highly conserved and specific regionsamong E. coli species and Shigella species. For example, within theEscherichieae family, gene probes defined by primers pairs ECM-1163,ECM-1958 (FIG. 4) and tor D-10574, tor D-11160 (FIG. 7) detect E. coliand Shigella species but not non-coli Escherichia species such as E.vulneris (ATCC 33821), E. hermannii (ATCC 33650) and E. blattae (ATCC29907). Gene probes defined by primer pairs 81B-7223, 81B-7794 (FIG. 12)and 81B-7278, 81B-7754 (FIG. 13) detect the presence of E. coli,Shigella flexneri and sonnei, except Shigella boydii. Gene probe definedby putP-5944 and putP-6693 (FIG. 19) detects E. coli, Shigella boydiiand Shigella sonnei and other enterobacteria (showing variable homology)but does not detect Shigella flexneri. Probe A detect E. coli andShigella species only (FIG. 2). This invention also indicates that whena large gene probe is reduced to many smaller gene probes, thespecificity of the smaller probes can be different from the parent probeand therefore must be tested out before use.

The method of the present invention encompasses the use of probes of anylength however it is envisages that the E. coli- and/or Shigellaspecies'-specific nucleotide sequences comprise at least 8 nucleotidesin length.

A further aspect of the present invention provides a molecular probecomprising at least 8 nucleotides obtainable from the nucleotidesequences of Formula I wherein said molecular probe is capable ofspecifically hybridizing to E. coli- and/or Shigella species'-derivednucleic acid molecules.

Still even yet more especially the present invention provides a methodfor testing and selecting other sequences in E. coli, Shigella speciesor related microorganisms in a sample, said method comprising subjectinga nucleic acid molecule preparation from said sample to genetic analysisusing one or more E. coli or Shigella species'-specific nucleotidesequences obtainable from one or more nucleotide sequences of Formula 1and/or Table 1 wherein the ability for said E. coli- or Shigellaspecies'-specific nucleotide sequences to hybridize to complementarynucleotide sequences in the nucleic acid preparation is indicative of anE. coli or Shigella species'-specific nucleotide sequence.

Some of the advantages of a smaller size probe are the ease with whichthey can be amplified by PCR to generate more material to work with andto carry out PCRISH.

The specificity of a smaller gene probe may be greater, equivalent orless. E. coli gene sequence at 7562 to 7794, AE000202 has relativelysimilar specificity as probe A (FIGS. 21, 20) to Enterobacter cloacaebut has the added advantage of not having homology to Shigella boydii(FIGS. 15, 2). A dilution assay indicates that Enterobacter cloacae DNAhas 50 times less hybridization signal intensity to both probe A and E.coli gene probe sequence (nt 7562 to 7794 of AE000202) as compared to E.coli DNA. Post hybridization wash condition is at 1×SSC, 0.1% w/v SDS,20 mM Na pyrophosphate at 65° C. Other examples such as those shown inFIGS. 3,4 and 7 indicate higher specificity than probe A. An example ofa more specific gene probe that recognizes E. coli and Shigella is thatdefined by primer sequence torD-10574 and torD-11160. Although the washcondition for FIG. 7 is at 0.1×SSC, 0.1% SDS, 20 mM pyrophosphate at 65°C., the probe is equally specific at 1×SSC wash (data not included). Thewash at 0.1×SSC indicates the hybrids are relatively stable andspecific.

DNA Probe Test, Selective Agar Media and Biochemical Test

The invention herein describes the specificity of the gene probe at nt7223 to 7794, AE000202 (FIG. 12), herein called 81B, as an example, inidentifying E. coli. This probe give 10 times less intense signal withEnterobacter cloacae DNA at the selected hybridization (see assaysection: hybridization and post hybridization conditions) and posthybridization wash (1×SSC at 65° C.) condition (FIG. 22). Despite thiscross over in hybridization signal with Enterobacter cloacae, the primersequence and gene probe sequence is relatively conserved. This molecularDNA test is compared to the selective isolation media and biochemicaltest. This comparative study is carried out on ATCC strains of E. coliand fecal isolates of subjects. The fecal isolates that are eitherpositive or negative for this gene probe are isolated and tested oneosin methylene blue (EMB) lactose-sucrose agar (bioMerieux, France),MacConkey agar (Oxoid, USA) and MUG agar (Oxoid, USA), API (bioMerieux,France) and sugar test.

All 66 (100% sensitive) randomly selected colony purified fecal bacteriaisolates that are positive for the probe 81B are identified to be E.coli with the API test strip and additional sugar test (4/66).

All 89 (100% specificity) randomly selected colony purified fecalbacteria isolates that are negative for the probe 81B are identified tobe either gram negative or gram positive microorganism but not E. colior Shigella species.

Of the patients' E. coli, 27 of 38 (71%) E. coli tested are EMBpositive, 56/66 (85%) of E. coli are tested positive on MacConkey andMUG agar plates alone. E. coli that is MacConkey positive need not beMUG positive and vice versa. The E. coli identified by the probe 81Bincluded lactose-negative E. coli which cannot be identified using theMPN method alone, or the MacConkey media alone or the MUG media alone.The probe detects the enteropathogenic serotype 0157:H7 and the 029:NME. coli that will not be picked up by the MUG assay. The selective agarplates and biochemical test are purchased in dried form andreconstituted according to supplier's instruction.

The 81B probe will recognize E. coli strains even those possessinghaemolytic properties, as observed on blood agar, and possessing DNAsequences that encodes known toxigenic genes, invasive genes, adherentgenes and cytonecrotizing genes.

One particularly preferred aspect of the present invention discloses amethod for detecting the presence of E. coli or Shigella species orrelated microorganisms in a sample as hereinbefore described whereinhybridization of E. coli- and/or Shigella species'-specific nucleotidesequences to the nucleic acid preparation is detected by the presence ofamplified nucleic acid products.

A further related aspect of the present invention provides a method fordetecting the presence of E. coli, Shigella species or relatedmicroorganisms in a sample wherein hybridization of E. coli- and/orShigella species'-specific nucleotide sequences to the nucleic acidpreparation or the presence of amplified nucleic acid products isdetected by a reporter molecule giving an identifiable signal.

Tissue Infection

Several techniques employing molecular hybridization for diagnosis ofbacteria infection can be used. They include southern filter or dot-blothybridization on DNA extracted from tissues. They may be useful but theyare unable to tell the investigator where precisely the infection is andif it is specific to certain cell type within a tissue. The DNAmolecular probes we have identified in this invention can be used forthose assays which follows the aforementioned approach as an earlyinvestigation tool. However, the use of primer sequences and DNA geneprobes can be made more informative in the technique ofpolymerase-chain-reaction-in-situ-hybridization (PCRISH). This techniquecan help in locating the presence of the microorganism, bearing in mindthat presence of specific antigen within a test sample, may notnecessarily imply that the identified pathogen is viable or necessarilycausative of the clinical infectious disease at that tissue site. Thecausative agent at the site of mucosal disease if isolated, identifiedand shown to have properties that can account for the diseased state,will provide additional evidence of its role for the disease condition.

The invention herein will describe the utility of the new probes to showthe association of bacteria DNA presence with histopathology of tissuesamples.

Presence of Bacteria in Tissue as Detected by PCR of Tissue TotalGenomic DNA

The invention herein used PCR technique in amplifying the gene sequence81B (primers: 81B-7223, 81B-7794) to test for its presence in total DNAextracted from colon tissue (total of 1 μg per 100 μl PCR reaction mix).Five microlitres of the PCR product mixture is resolved on ethidiumbromide stained agarose that has a molecular weight marker on one of thetracks. A PCR product migrating at the expected position and ishybridization positive for the ³²P labeled DNA probe (probe sequencecorrespond to nucleotide (nt) 7278 to 7773 of AE000202) is taken aspositive. The inventors found the presence of the sequence as visualizedunder 312 nm ultra-violet (uv) illumination, in all cancer patientsstudied, either in tumour tissues (25/29, 86%) or in adjacent normaltissues (17/23, 74%). In contrast, in 34 control patients with nocancer, but is admitted for some related gastrointestinal complaints,this sequence is found in 7/34 (21%). The P value is <0.001.

It is emphasized that confirmation of target PCR product can be carriedout by hybridization of the Southern transferred membrane bound PCRproducts to specific radiolabeled gene probes.

This data suggests that colon tissue from patients with colon cancerharbor more bacteria than patients with no colon cancer. Such data areinformative when correlated with histology, clinical diagnosis andpathogenic bacteria isolates from tissue samples devoid of contaminants.

Accordingly, yet a further related aspect of the present inventionprovides a method for detecting the presence of E. coli, Shigellaspecies or related microorganisms in a sample wherein the samplecomprises food, water, semi-solids or semi-liquid material, mammaliantissue, tissue extract or cells of tissue or normal tissue or tissuepredisposed to cancer growth or malignancy or cellular instability.

In a particularly preferred aspect of the present the mammalian tissueis associated with colon, stomach or colorectal tissue.

A related aspect of the present invention provides a method foridentifying nucleotide sequences, or their expressed products, capableof inducing or otherwise facilitating abnormal cell growth or abnormalphysiology, said method comprising introducing a nucleotide sequencecomprising E. coli- and/or Shigella species'-specific nucleotidesequences from the nucleotide sequences in Formula I into cells andobserving morphological and/or physiological changes to said cellscompared to control cells without said introduced nucleotide sequenceswherein the presence of abnormal morphology and/or physiology in a cellis indicative of a nucleotide sequence from Formula I, or a polypeptideexpressed therefrom, which is capable of inducing or facilitatingabnormal cell growth or physiology.

In a further preferred aspect of the instant invention the abnormal cellgrowth or physiology is associated with cancer or a predisposition tothe development of cancer or cellular instability.

The genes encoded within Formula I can be transcribed into mRNA andtranslated into functional proteins. These can serve as targets fordetection and intervention. The detection systems that can be usedinclude imaging, scintigraphy, immunohistological methods,enzyme-chemical/amplification detection methods, chemical methods andmicrochip computer assisted methods (direct/indirect). These can becarried out on patients or animals or on samples such as serum, tissues,food and liquids.

The invention can be used in the following areas:

-   (1) Molecular diagnosis—for example, testing patients, e.g.    predisposed to cancer, cancer, having infection, for the presence of    Formula I and/or its transcripts;-   (2) Serological diagnosis—for example, testing patients predisposed    to cancer, cancer having infection, for the presence of the protein    encoded by the Formula I sequence, or that specifically binds to    such a polynucleotide, or testing such patients for the presence of    antibodies to such protein. A sample from the patient is preferably    blood, urine or other body fluids, tissue or excretion products;-   (3) Immunohistochemistry/histochemistry applications—for the    diagnosis of predisposition to cancer, cancer, cellular instability,    infection in tissue samples;-   (4) Diagnostic imaging—in which case the antibody or probe will have    an appropriate label or marker, for example, a radioactive label or    marker;-   (5) Therapy    -   (a) for example, antibodies of the present invention may form        part of an immunotoxin, in order to deliver toxic agents, such        as plant toxins, e.g. ricin, to the site of a malignant or even        a benign tumour (see, for example, European Patent Application        No. 84304801.8—Publication No. 0145111);    -   (b) for example, polynucleotides of the present invention may be        useful alone in therapy as anti-sense DNA or RNA. Thus,        polynucleotides of the present invention optionally in a vector        or in a polynucleotide analogue, which contains sequences        complementary to DNA or RNA defining a protein which is        differentially expressed during cancer initiation, progression        and metastasis or a portion thereof can be employed to prevent        expression of the said protein;    -   (c) for example, the metabolic pathway that the Formula I        sequence or its nearby sequence encode can be utilized to        activate pro-drugs;-   (6) Histological analysis—DNA or RNA or protein having an    appropriate label or marker may be useful for in-situ detection for    histological analysis; and-   (7) Food and water sanitation.    Polymerase-Chain-Reaction-In-Situ-Hybridization Technology (PCRISH).

In this invention herein, we will describe the new use for DNA probesthat permit the detection and localization of members of Escherichieaefamily (and Shigella species) in paraffin embedded tissue samples bypolymerase-chain-reaction-in-situ-hybridization (PCRISH). This is usefulfor the study of (1) clinical conditions with bacteria infection (2)treatment and infection (3) association of bacteria in the developmentof cancer and (4) identifying cells at risk of or having genome that isgenetically unstable.

These investigations can be carried out on routinely processed,formalin-fixed and paraffin-embedded tissues obtained from pathologyfiles at any hospital. Such tissues have excellent preservation oftissue architecture and cellular detail and allow retrospective analysisof stored tissue blocks. To illustrate the utility of theabove-mentioned probe, we will provide data on its use in the study ofcolon and gastric related conditions.

Sensitivity and Specificity

This invention herein will provide data on how sensitivity andspecificity of an assay can be dramatically improved by carrying out ahybridization step after PCR. The inventors will describe an assay thatwill simulate PCRISH in-vitro. First, PCR is carried out on randomlyselected E. coli (number, N=3), Shigella sonnei (N=1), Enterobacteragglomerans (N=1), Pseudomonas aeruginosa (N−1), H. pylori (N=1) frompatients, TG2, K12 and the panel of ATCC bacteria total genomic DNA(Table 3). A given volume of the PCR product mixture is then resolved onan agarose gel, and the PCR products visualized by ethidium bromidestaining under UV illumination. The primer targeted bacteria sequencewill give a single band. Many additional bands indicates the lack ofspecificity of the primer sequences. The products in the gel aresubsequently transferred onto nylon-plus membrane (Amersham PharmaciaBiotech Inc, USA) by the method of Southern, and hybridized to ³²Prandom-prime labeled sequence specific probes. This hybridization stepincreases the detection level and also improves the specificity of theassay by hybridizing specifically to homologous sequences. Specificityis denoted by a single band when exposed to an autoradiograph. Suchassays indicate whether the primers and probes are specific and can helpto detect the presence of bacteria when used in PCRISH. The selection ofprimer and probe sequence within the Formula 1 can be empiricallycarried out in a step-wise systematic fashion until all the appropriateones are selected.

Gene sequences between nt 7419 and 7985 (AE000202), herein called theOH, the 81B (nt 7223 to 7794, AE000202) and the H. pylori ribosomal gene(Table 3) have been tested in accordance with the present invention. Byvarying the PCR conditions, OH-7419 and OH-7985 primers directed PCRamplification of bacteria DNA could give rise to specific andnon-specific products. Any DNA sample giving rise to PCR amplified band,whether or not in the presence of non-specific bands, and migrating inagarose gel at the expected molecular weight is denoted as positive(Table 3). Hybridization of labeled gene probe defined by the sequencebetween primers OH-7562 and 81B-7794 specifically picks up PCR productsfrom E. coli and Shigella species except Shigella boydii. Primers81B-7223 and 81B-7794 are more specific under the PCR conditions usedand give specific bands of expected molecular weight. The labeled geneprobe having the sequence between nt 7278 and 7773 specificallyhybridizes to the products from E. coli and Shigella species exceptShigella boydii. The specificity of this latter gene probe for genomicDNA of E. coli species is shown in FIG. 23. Although there is lowhomology between Enterobacter cloacae DNA and OH gene probes (low, FIGS.14,15,21) and 81B gene probes (relatively higher than OH, FIGS.12,13,22), the aforementioned OH and 81B PCR primer pairs do not amplifyspecific products from Enterobacter cloacae DNA.

The dramatic increase in sensitivity and specificity is illustrated bythe H. pylori ribosomal gene primers and gene probe. FIGS. 24 and 25shows that the H. pylori probe has a low to moderate level of homologyto enterobacteria and gram positive bacteria DNA even with stringentpost hybridization wash condition of 0.1×SSC, 0.1% SDS, 20 mMpyrophosphate at 65° C. However, upon PCR and hybridization to DNA of aselected number of enterobacteria and gram positive bacteria (Table 2a,grid D) likely to be found in the gastrointestinal tract, only H. pyloriPresence is detected (Table 3). FIG. 26 and Table 2a, grid F will showthe dot-blot hybridization result where the detection limits for thepresence of H. pylori DNA is dramatically improved after a simulatedPCRISH technique. Two hundred nanogram each of total genomic HP DNA anddifferent purified fecal isolates of E. coli genomic DNA from differentpatients, are each PCR amplified in 100 μl of reaction mixture. AfterPCR, 10 μl is taken out from the H. pylori reaction tube and dilutedwith 90 μl of water before a known volume is being dot-blotted ontoAmersham nylon plus membranes and hybridized to random primed ³²Plabeled H. pylori (HP) ribosomal probe.

FIG. 26, column A, rows 1 to 4 each contained 1, 5, 10 and 50 μl of the10 fold diluted H. pylori post PCR reaction mixture. The hybridizationsignals intensified with increase in amount of PCR products. Rows 5 to 8contained 1, 5, 10 and 50 μl of 10 fold diluted PCR product comprisingof equal portions of 4 E. coli DNA PCR products (K12, 142/31, 179/36,197/5 and 117/3B). No amplified PCR products are obtained with H. pyloriprimer directed amplification of E. coli DNA. This column shows that theH. pylori primers are specific for the H. pylori genome.

FIG. 26, column B, rows 1 to 5 contained mixture of 5 μl diluted post H.pylori PCR reaction mixture and 5 μl (undiluted) of post PCR reactionmixture of different E. coli DNA isolates respectively. The resultindicates that the hybridization of HP probe to the H. pylori PCRproducts is not affected by the presence of E. coli DNA. This isindicated by the equal intensity of the dot.

FIG. 26, column C, rows 1 to 5, each contained 5 μl (undiluted) post PCRreaction mixture of different E. coli isolates of different patients.The HP primers did not PCR amplify E. coli DNA.

FIG. 26, rows 6 to 8 of column B, each contained 10 ng, 50 ng and 200 ngrespectively of H. pylori genomic DNA and in column C, each containedinstead a respective equivalent of E. coli genomic DNA. The HP probeshowed higher homology to its own gene.

Hybridization of a panel of bacterial DNA to the HP gene probe has shownthat it has some homology to the other bacteria species (FIGS. 24, 25).However by combining sequence specific primer directed PCR andhybridization with a probe that has low homology to other species, it isstill possible to increase the sensitivity and specificity of HPdetection (FIG. 26, Table 2a- grid F).

Exemplification of various aspects of the invention using PCRISH in noway limits the methods of the present invention and any appropriatetechnique may be used to monitor or detect the presence of E. coli DNAin cells. This invention herein relates the use of PCRISH as a methodfor illustrating how pathogens can be detected in-situ. It does notexclude the use of in-situ hybridization nor in-situ-PCR technology,using sequences within Formula I, as an alternative. The amount oftarget, reporter system, PCR cycles and hybridization conditions areimportant determinants in deciding which method is appropriate.

Bacteria Detection in Tissue Specimens Using PCRISH Technology

A preferred method in amplifying vast amount of DNA sequences incells/tissue and detecting the presence of the sequences in-situ use thePCR-in-situ hybridization (PCRISH) technology. PCRISH is practicedroutinely by those having ordinary skill in the art and its uses indetecting infection in-situ is widely used and accepted.

As a start, cells or tissues are fixed with a suitable fixative (example10% buffered formalin) for 2–72 hrs and the tissues subsequentlyparaffin-embedded. The paraffin-embedded tissues are then sectioned at3–4 microns and mounted on aminoalkysilane treated glass slides. Beforeamplification is undertaken, the sections are deparaffinized first andthen carefully permeabilized using proteolytic enzymes such asproteinase K, so that the cellular morphology is maintained and the DNAis accessible. The cell/tissue is now ready to permit access of PCRreagents into the cellular compartment where the nucleic acids arefound. Amplification then occurs at the defined target intracellularsequence. Detection of amplified product is then carried out byhybridizing with a non-radioactive labeled DNA probe. We used DNA probeslabeled with Digoxigenin-labeled nucleotides. This DIG-labeled probe isdetected with anti-digoxigenin (anti-DIG) antibodies that are conjugatedto alkaline phosphatase. This alkaline phosphatase is visualized withcolorimetric (NBT and BCIP) alkaline phosphatase substrates (BoehringerMannheim). It is imperative to always to perform a negative control witheach specimen and to understand that a negative result only indicates aninability to detect the substance analyzed. Negative tissue control doesnot contain the specific antigen in question. Positive control mustaccompany each run of test tissue sections within which the specific DNAsequence is sought.

Interpretation of Hybridized Tissue Sections.

It is known that some members of the Escherichieae family (E. coli andShigella species) are able to adhere to, invade cells and move acrosscells. Therefore, there is the need to correlate histopathology withquantity and depth of bacteria presence in order to evaluate their rolein gastrointestinal infection and cancer. This is done by studying thequantity and spatial distribution within the tissues of thehybridization signals generated with the DIG-labeled bacteria DNAprobes.

We have categorically defined the spatial distribution of the bacterialpresence into positive and negative depending on whether the signals arefound (i) outside mucosa cells/lumina propria (negative), (ii) in mucosacells (positive) and (iii) in nuclei of mucosa cells (positive). Alltissues are obtained upon discovery (pre-treatment) unless statedotherwise.

Colon

The use of primer and probe DNA gene sequence of 81B for carrying outPCRISH in colonic mucosa tissue in this invention is characteristicallyfound to identify all hyperplastic tissues (number [N]=7; FIG. 27),adenomas (N=12; FIG. 28), adenocarcinomas (N=11; FIG. 29) and metastaticcolonic cells in liver specimens (N=5; FIG. 30) to be positive for the81B marker probe. The signals are characteristically predominant incells and in nuclei of the cells. The hyperplastic tissues, adenomas andadenocarcinomas can also have 81B signals located outside cells.Metastatic cells, with primary in the colon, found in liver specimensshow distinct 81B signals in the nucleus and without significantexogenous 81B signals outside cells. Mucosa tissues of normal histologyobtained adjacent to tumours (N=8; FIG. 31) are positive in certainareas and have intense signals in those areas. The majority of tissueswithout cancer (N=16 ; FIG. 32) and obtained from normal patientsindicated for colonoscopy are negative (69%). One example of an infectedtissue is that of a patient's colon tissue diagnosed to have proctitison histology (FIG. 33). Here, the distribution of 81B signal isdiffusely distributed but without the intense cellular/nuclear signalthat is observed in normal tissue adjacent to tumours (FIG. 31) and intumours (benign, pre-malignant and malignant).

H. pylori Probe is insignificant in metastatic cells in liver sections(N=5; FIG. 34) and serves as a negative control.

The transition from normal through pre-malignant to localized malignantand finally a metastatic stage has been histologically defined forcolorectal cancer. Therefore the appearance of 81B signals at earlystages and before histological changes, through to all histologicalchanges associated with cancer and leading to the metastatic stageindicates a possible aetiological role for E. coli/Shigella. Thisinvention herein identifies several uses for the E. coli/Shigella probewhereby markers specific for E. coli/Shigella can be used for screeningpatients for risk of colorectal cancer, colorectal cancer and infectionwith E. coli/Shigella.

Gastric

Helicobacter pylori is established as the major aetiological agent ingastritis and peptic ulcer and it is also known to infect half of theworld's population. This high infection/disease ratio is howeverexplained by host factors, socio-economic conditions of the variouscountries and infection by a sub-population of virulent Helicobacterpylori (Chu, K. M. and Branicki, F. J. JAMA SEA 13:5–7, 1997).Therefore, it is clear that to understand the pathogenesis ofHelicobacter pylori and general infection, one approach is to preciselylocate the bacteria to the site of mucosal disease and to correlate itto histology and clinical presentation. Past data usingimmunocytochemical detection system (Cartun et al. J Clin Pathol.43:518. 1990) and in-situ hybridization technique have both localizedthe bacteria to the luminal, foveolar epithelium or periepithelialmucous layers of the gastric mucosa of helicobacter pylori associatedgastritis (Van Den Berg et al. J Clin Pathol. 42:995–1000, 1989; Bashiret al. J Clin Pathol 47:862–866, 1994). Unlike these observations, weare able to show that Helicobacter Pylori can be found beyond theluminal or foveolar epithelium in patients with gastritis and may assistin understanding the significance of high infection/disease ratio. Thisinvention will highlight the resolution power of a successful PCRISHtechnique for studying the association of endoscopic diagnosis ofgastric conditions with histological findings, bacteria pathogenesis andclinical presentations.

The invention herein has found E. coli, other than H. pylori, in stomachrelated conditions such as gastritis of varying severity, ulcer andcancer.

The use of primer and probe DNA gene sequence of H. pylori and 81B forcarrying out PCRISH in stomach tissue in this invention is found toidentify all H. pylori Positive active chronic gastritis (N=9), H.pylori positive gastritis (acute on chronic) (N=12), active chronicgastritis with no histologically detectable H. pylori (N=10), chronicgastritis with no histologically detectable H. pylori (N=9), mildgastritis with no histologically detectable H. pylori (N=4), normal withno histologically detectable H. pylori (N=4), normal tissue distant totumour (N=1), adenocarcinoma (N=5) and liver specimen with poorlydifferentiated metastatic stomach adenocarcinoma (N=1), positive for the81B sequence. The distribution of the 81B sequence is such that they canbe in the luminal areas with variable degree of penetration intointercellular, cellular and nuclear areas of specimens. The area ofinfection is patchy. For the cancer specimens, the signals are foundmainly in the nuclei of tumours (FIG. 35) and in normal tissue adjacentto tumours (FIG. 36) they are found both in the nuclear and luminalarea. For the metastatic cells in liver section, that originated fromthe cancer at the gastro-oesophageal junction, the signals are alsomainly. in the nuclei but without the exogenous signals that cansometimes be observed with the primary tumour (FIG. 37). Gastric mucosawith normal histology and no histologically detectable H. pylori butpositive for the 81B probe is interesting as the patient presented withnon-ulcer dyspepsia, epigastric discomfort and vomiting. The 81B signalsshow patchy aggregates. FIGS. 38 and 39 are of the same specimen butshowing different area

The samples that are histologically diagnosed to be positive for H.pylori are confirmed by PCRISH. Although the H. pylori probe is foundpresent in normal tissue distant to the adenocarcinoma of stomach (FIG.40), it showed insignificant nuclear signals in the tumour itself (FIG.41). H. pylori probe is not detected in metastatic tumour cells in liver(FIG. 42).

This invention herein presents a use for the E. coli/Shigella probes ingastric related studies whereby it can be used to screen for patientswith risk of gastric cancer, gastric cancer and gastric infection. Whenfound in the presence of H. pylori, it supports the possibility thatsynergistic interaction between two types of bacteria can be a cause forgastric related conditions (active chronic gastritis, FIGS. 43, 44). Howimportant the presence of H. pylori is to E. coli is yet to bedetermined as it is known that E. coli has a mechanism for acidresistance. (Lin J, et al. Applied and Environmental Microbiology62:3094–3100, 1996). In addition, E. coli are known to have invasive,cytonecrotizing, adherent, and toxigenic genes that can account forgastric related conditions.

Cellular Instability

Studies have suggested that apparently uninvolved mucosa adjacent to,and even remotely from colorectal cancer is abnormal morphologically andhistochemically (Ngoi et al. Cancer 66:953–958, 1996). In addition, theinvention herein showed E. coli/Shigella DNA marker in the nucleus ofmany normal cells next to tumours, tumour cells and metastatic tumour.This suggests that E. coli/Shigella probe can be a marker associatedwith risk of cellular instability or cellular instability andtumourigenesis as it is known that bacteria DNA has tumourigenicpotential.

Isolation of Microorganism that is Associated with Tissue Infection

The invention herein indicates a method whereby in addition tolocalization of bacteria in tissue sample, the marker sequence canfurther assist in identifying and isolating members/strains of thefamily of bacteria it represents, in tissue/fecal specimens.

To start off at the tissue level, using PCRISH, the spatial distributionof the marker (for example, 81B or OH or any other that is within theFormula 1 sequence) would indicate whether there is a pathogen present.This observation may be supported by histological diagnosis forinfection, benign, pre-malignant or malignant cancer. This is thenfollowed by detecting for positive colonies isolated from the positivespecimens (for example tissue, fecal), and hybridizing to the positivecolonies, separately, a panel of DNA that is known to encode eitherinvasive, adherent, necrotizing, toxigenic or other pathogenicproperties. The bacteria that is found to possess pathogenic propertiesas defined by a DNA sequence, can have their presence within the tissuechecked by PCRISH using primers and hybridizing probes for thatproperty. From hereon, association studies can be carried outempirically with the various pathogenic probes to understand bacteriapresence and infection, cancer risk and etiology of cancers.

With this approach, the inventors have found pathogenic E. coli to bethe main pathogen associated with cancer risk and cancer. Herein theinventors define pathogenic E. coli as those strains of E. coli able toinvade into cells and those that can cause cellular changes in cells.Since pathogenic E. coli is found in the instant invention as a markerof cellular instability and tissue at risk of cancer (benign tumours,pre-malignant tumours) and cancers (malignant tumour/cells), ittherefore implies that any sequence of E. coli (pathogenic or otherwise)can be used for the above purpose.

A related aspect of the instant invention provides a molecular probe ofat least 8 nucleotides, identified by the methods as herein describedwherein said probe comprises a sequence of nucleotides from Formula Iand wherein said molecular probe is capable of specifically hybridizingto E. coli and/or Shigella species'-derived nucleic acids.

Assays

Microbiological techniques are practiced routinely by those havingordinary skill in the art and its use is wide and accepted. Informationfor practicing is disclosed herein by reference: “Nga, B. H. and Lee, Y.K.: Microbiology Applications in Food Biotechnology. Elsevier AppliedScience, 1990. Koneman et al. Introduction to Diagnostic Microbiology. JB Lippincott Company 1994. AOAC Official Methods of Analysis 1995.Pepper I L., Gerba C P and Brendecke J W: Environmental Microbiology: Alaboratory Manual. Academic Press 1995. P R Hunter: Waterborne Disease:Epidemiology and Ecology. John Wiley and Sons 1997.

DNA and related technology is widely used by those having ordinary skillin the art and information for practicing DNA and related technology isdisclosed herein by reference: Moseley et al. The Journal of InfectiousDiseases 142:892–898, 1980. Berger S L and Kimmel A R: Guide toMolecular Cloning Techniques. Methods in Enzymology. Volume 152.Academic Press Inc 1987. Tenover F C: DNA Probes for InfectiousDiseases. CRC Press 1989. Sambrook J, Fritsch E F and Maniatis. T:Molecular Cloning: A laboratory manual. Second Edition. Cold SpringHarbor Laboratory Press 1989 (3 volumes); Echeverria et al. J ClinicalMicrobiol 27: 31–334, 1989; Virginia L C and Bavoil P M: BacteriaPathogenesis. Academic Press 1997.

Aspects of the present invention include various methods such as PCR anddot-blot, for example, for determining whether a sample containsbacteria sequence of the Escherichieae family by molecular analysis.Nucleic acid-based assays can be made exquisitely sensitive, and theirspecificity can be adjusted from broad to a very narrow range by carefuldesign of the probe and precise control of hybridization conditions.

The invention relates to Formula I, DNA gene probes and primers used inthe method of identifying DNA that is encoded in the DNA of members ofthe Escherichieae family such as the E. coli and Shigella species.

The DNA sequence based methods for determining whether a sample DNAencoding the sequence found in E. coli and Shigella species include butare not limited to polymerase chain reaction technology, Northern andSouthern blot technology, dot-blot, colony-blot, PCR-in-situhybridization, in-situ-hybridization technology and oligonucleotidehybridization technology.

The invention primarily involves methods in DNA detection and thesemethods are commonly used alone or in combination so as to enhance thesensitivity and specificity of the DNA sequence for detecting thepresence of species of E. coli and Shigella.

It is contemplated that other sequence-based methodology for detectingthe presence of bacteria DNA in samples may be employed according to theinvention.

PCR Technology

A preferred method in amplifying vast amount of DNA sequences uses thepolymerase chain reaction (PCR) technology. PCR technology is practicedroutinely by those having ordinary skill in the art and its uses is wideand accepted. Methods for practicing PCR technology are disclosed in“McPherson M J, Quirke P and Taylor G R: PCR. A Practical Approach.Volume 1. Oxford University Press 1994”, which is incorporated herein byreference.

The nucleotide sequence present in species of E. coli and Shigella iswell known such as in GenBank database accession number AE000201,AE000202 and AE000203. To perform this method, DNA is extracted/releasedfrom cells in a sample and tested using well-known methods and readilyavailable starting material.

The DNA is combined with the primers, free nucleotides and enzymefollowing published PCR protocols. The mixture undergoes a series oftemperature changes. If the DNA encoding the marker sequence for E. coliand Shigella species are present, that is, if both primers hybridize tosequences on the same molecule, the molecule comprising the primers andthe intervening complementary sequences will be exponentially amplified.The amplified DNA can be easily detected by a variety of well knownmeans. If the sequence is not present, no DNA molecule will beexponentially amplified. The PCR technology therefore provides anextremely easy, straightforward and reliable method of detecting E. coliand Shigella species DNA in a sample. Publication is herein provided byreference for indicating how PCR technology is utilized for detectingpathogens in food, Gannon et al Applied and Environmental Microbiology58:3809–3815, 1992; AOAC Official Methods of Analysis 1995.

PCR Primers

PCR primers can be designed routinely by those having ordinary skill inthe art using well known DNA sequence information that can be retrievedfrom GenBank database. Software programs are also available to assist indesigning optimal primer sequences as long as sequences of gene ofinterest are known. (PCR PLAN (PC/Gene system, Intelligenetics, inc.USA). Primers are generally 8–50 nucleotides, preferably 18–28nucleotides. A pair of primers is routinely used for PCR. Whenperforming PCR on extracted DNA samples containing targeted specificbacteria DNA, multiple copies of the DNA will be made. If the targetedDNA is not present, PCR will not generate a discrete detectable product.

PCR Product Detection

PCR amplified DNA may be detected by several well-known methods. Thepreferred method for detecting the presence of amplified DNA is toresolve the PCR reaction material by gel electrophoresis and stainingthe gel with ethidium bromide in order to see the amplified DNA ifpresent. A standard molecular weight marker containing a standard ofequivalent size to the expected size of the amplified DNA is preferablyrun on the gel as a control.

In some instances, such as when small amounts of amplified DNA isgenerated at the first attempt, therefrom, it is desirable or necessaryto perform a PCR reaction on the first PCR reaction product. A nestedset of primers is used in the second PCR reaction. The nested set ofprimers can either hybridize to sequences downstream of the 5′primer andupstream of the 3′primer used in the first reaction or hybridize furtherinto the first PCR product and allow a second PCR reaction.

The present invention includes oligonucleotide sequences, which areuseful as primers for performing PCR methods to amplify DNA fragmentsthat are useful for identifying the Escherichieae family in particularE. coli and Shigella species. For the detection of the sequence,labeling of DNA can either be radioactive or non-radioactivenucleotides.

Random Prime Labeling of DNA Fragment (Megaprime DNA, AmershanmPharmacia Biotech Inc, USA)

Labeling can be carried out according to manufacturer's recommendation.Between 100–500 ng of DNA fragment is heated for 5 min with optimizedamount of hexamer primer-solution (7 μl) before cooling rapidly to roomtemperature. This is then mixed with reaction mixture containing 12 μlof labeling buffer, 5 μl enzyme (1U/μl), 5 μl of ³²P (6,000 Ci/mmol) andwater to 50 μl final reaction volume. Labeling is carried out at 37° C.for 3 hrs. Separation of unincorporated ³²P from labeled sequence iscarried out by spin column method (Biospin columns: Bio-RadLaboratories, USA).

The amount of sequence to be labeled can vary over a wide range as otherparameters can influence the outcome of the invention. Examples toconsider: the efficiency of the labeling system employed as othersystems of labeling are available (polymerase chain reaction [PCR]labeling and use of radioactive or non-radioactive nucleotides), thenature of the sequence, either as probe A or in combination with othersequences or sub-fragments of the Formula I sequence.

PCR on Total Genomic DNA.

One hundred microlitres of PCR reaction mixture containing 10 mM KCl, 10mM(NH₄)₂SO₄, 20 mM Tris HCl (pH 8.0), 2 mM MgSO₄, 0.1% Triton X-100, 200μM (each) of dNTPs, 0.4 μM (each) of two primers, 1.5 unit of Taqpolymerase and 1 μg of total genomic DNA (e.g. extracted from colontissue) is amplified in a thermal cycler (Perkin Elmer Cetus, modelTC1). Amplification is carried out in 3 stages: (i) 94° C. for 3 min;(ii) 10 cycles of 30 s at 94° C., 30 s at 62° C., 1 min 30 s at 72° C.,and then (iii) 35 cycles of 1 min at 92° C., 40 s at 58° C., 1 min 30 sat 72° C. with an auto extension of 5 s at 72° C. at the end of eachcycle. Lastly, an extension time of 7 min at 72° C.

Hybridization and Post Hybridization Conditions

The complete/partial sequence of formula I either alone or as a mixtureare radioactively labeled by random-prime method using ³²P-dCTP(alpha-³²P-dCTP, 6,000 Ci/mmol, Amersham Pharmacia Biotech Inc, USA).Nylon membranes (Hybond-N+, Amersham Pharmacia Biotech) arepre-hybridized overnight at 65° C. in a buffer containing 5×SSC (1×SSCis 0.15 M sodium chloride, 0.015 M sodium citrate) 0.1% SDS, 5×Denhardt's solution (1× Denhardt's solution is 0.02% bovine serumalbumin, 0.02% polyvinyl pyrolidine 360K MW and 0.02% ficoll 400K MW inwater), 200 μg/ml sonicated denatured salmon sperm DNA (sssDNA).Hybridization is carried out at 40° C. for overnight in a buffercontaining 5×SSC, 20 mM sodium phosphate, pH 7.0, 1×Denhardt's solution,0.1% SDS, 200 μg/ml sssDNA, 25% deionized formamide and labelled probe.The probe used can be from 100 to 500 ng of labelled sequence. Themembranes are washed at 65° C. in 5×SSC, 0.1% w/v SDS, 20 mM Napyrophosphate for several washes and followed by another few washes at65° C. with 1×SSC, 0.1% w/v SDS and 20 mM Na pyrophosphate. All washesranged from 30 to 60 min or until the background signal is acceptablylow. Autoradiographs are exposed overnight at −70° C.

Several parameters can be empirically adjusted to improve on thespecificity of the hybridization assay. An increased in stringency ofthe assay can be achieved by increasing the percentage formamide usedduring hybridization and/or by decreasing the salt concentration of thepost hybridization wash buffer. Use of alpha-³²P-dCTP, 3,000 Ci/mmol ispossible as this will only affect the level of sensitivity of detection.

Polymerase-Chain-Reaction-In-Situ-Hybridization Technology

Incorporated herein are references that are useful for samplepreparation and understanding the principals and application of PCRISH:“Rentrop et al. Histochemical J 18:271–276, 1986, Carson, F. L.Histotechnology. A Self-Instructional Text. American Society of ClinicalPathologists Press 1990; Mikel U V: Advanced Laboratory Methods inHistology and Pathology. Armed Forces Institute of Pathology 1994;Nuovo, G. J.: PCR In Situ Hybridization. Protocols and Applications.Raven Press, N.Y. 1994; Gu, J: In Situ Polymerase Chain Reaction andRelated Technology. Eaton Publishing Co. USA 1995; Boehringer MannheimNon-radioactive It Situ Hybridization Application Manual (2^(nd)edition) Washington, D.C., 1996; O'Leary et al. J Pathol. 178:11–20,1996.

Fixation and Tissue Preparation

Pathology departments routinely fix tissue that are targeted forembedding in paraffin wax and which are subsequently used for diagnosisof tissue pathology. For fixation, we used for our biopsy specimens, 10%buffered formalin for 48–72 hrs (100% formalin diluted in phosphatebuffered saline [PBS] to a 10% v/v concentration). After paraffinembedding, specimens of 3–4 micron thickness are cut onto3-aminopropyltriethoxysilane (silane) coated microscope slides.Requirement of tissue thickness is dependent on the overall size of thetissue to be tested. The inventors recommend 3 micron for biopsyspecimen and 4 micron for larger specimens. Before the samples can beused for PCRISH, the sections are fixed on slides at 58° C. for 1 hr 20min, followed by deparaffinization in xylene (3×5 min) and final rinsein 100% (3×1 min) ethanol. At this stage, the sections can be left foruse the following day. Just prior to use, the sections are rehydrated 2min each through graded (70% and 50%) ethanol and water with finalequilibration in PBS for 5 min.

PCR Labeling

PCR labeling of probes for PCRISH is carried out using digoxigenin dUTP(Boehringer Mannheim). This is carried out in a 100 μl reaction mixturecomprising of 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris HCl (pH 8.0). 2 mMMgSO4, 0.1% Trition X-100, 50 μM DIG-11-dUTP, 150 μM dTTP, 200 μM eachof dATP, dCTP, dGTP, 0.4 μM primers each, 2 units of Taq DNA polymerase(Perkin Elmer, UK) and 2 μl of first PCR product. The first PCR productis made with the outer primers for the same gene using K12 bacteriagenomic DNA.

Amplification is carried out in 4 stages in a thermal cycle (GeneAmp PCRSystem 2400 or 9600, Perkin Elmer, UK):

(i) 94° C. for 2 min, (ii) 5 cycles of 30 s at 94° C., 30s at 58° C.,90s at 72° C. (iii) 10 cycles of 30 s at 94° C., 30s at 55° C., 90 s at72° C. and (iv) 30 cycles of 60 s at 92° C., 30 s at 52° C. 90 s at 72°C., with an autoextension of 5 sec at 72° C. at the end of each cycle.Final extension time is 7 min at 72° C.

Proteinase K Digestion

For optimal cell permeabilization, each tissue type is empiricallytitrated against time at 40° C. for any given concentration ofproteinase K. The inventors routinely use a fixed concentration of 10μg/ml of proteinase K in PBS and adjust the time of digestion. Theenzyme is diluted from a 10 mg/ml stock into the 40° C. pre-warmed PBSsolution for 15 min before starting the permeabilization step. The size,type of tissue (normal or fibrous and necrotic) and how long it has beenarchived affect the duration of proteinase K digestion. After digestion,the sections are rinsed several times in PBS for a total of 5 min tostop the proteinase K digestion. This is followed by incubating in 10%buffered formalin for 1 min at room temperature and a further rinse inseveral changes of PBS to remove excess formalin. The sections are thendehydrated by incubating them, for 3 mins each, in a graded ethanolseries of 70%, 85% and 100%. The slides are air dried at roomtemperature and free of ethanol before PCR.

In-Situ PCR

Twenty five microlitres of PCR reaction mixture containing 10 mM KCl, 10mM (NH₄)₂SO₄, 20 mM Tris HCl (pH 8.0), 2 mM MgSO₄, 0.1% Triton X-100,200 μM (each) of dNTPs, 0.6 μM (each) of two primers, 1 unit of Taqpolymerase is overlayed onto each tissue section. Glass coverslip (24×40mm) are then carefully put on top, taking care not to create airbubbles. The cover slips are sealed on all sides with nail vanish, thenair dried before placing the slides into the thermal cycler (GeneAmp Insitu PCR System 1000, Perkin Elmer Cetus). Amplification is carried outin 3 stages: (i) 94° C. for 2 min; (ii) 10 cycles of 30 sec at 94° C.,30 sec at 58° C., 60 sec at 72° C., and then (iii) 15 cycles of 30 secat 92° C., 30 sec at 55° C., 60 sec at 72° C. with an auto extension of5 sec at 72° C. at the end of each cycle. Lastly, an extension time of 7mins at 72° C.

Post PCR Wash

After PCR, a check for air bubbles inside the cover-slip is always made,and its location and size is recorded before incubating them 5 min eachat 92% and 100% ethanol respectively. The validity/interpretation of thefinal data will take into account the presence or absence, size andlocation of the bubble. Bubbles create artifacts. The layer of varnishis carefully peeled off the coverslip and the sections rinsed severaltimes with 2×SSC before soaking twice in 0.5×SSC at room temperature for5 min each. It is important that the varnish does not touch thespecimen. The section is then treated with 10 μg/ml proteinase Ksolution for approximately 15 s before rinsing several times with PBSand dehydrated through a graded ethanol series (1 minutes each in 70%,85% and 100% ethanol). The slides are air dried at room temperaturebefore hybridization.

Hybridization

Twenty five microlitre of hybridization buffer (comprising of 25%formamide, 1×dendhardt solution, 5% (w/v) dextran sulphate, 200 μg/mlSSS DNA, 4×SSC and 5% (v/v) DNA probe) is overlaid onto the tissuesection. Coverslip is placed over each tissue section, taking care notto create air bubbles. Coverslip must be larger that the specimen, witha minimum distance of 3 mm away from all sides of the tissue. Thecoverslip is sealed on all sides with rubber cement and the cement isthoroughly air dried before the denaturation step. The sections areplaced over a 95° C. heating block for 6 min to denature the DNA,rapidly cooled on ice for 1 min and finally incubated overnight at 40°C. in a humid chamber.

Post Hybridization Wash

After the overnight incubation, the rubber cement is carefully peeledoff and the section rinsed several times in 0.5×SSC, twice for 5 min in0.5×SSC at room temperature, and in 0.1×SSC at 42° C. for 20 min (2×5min, followed by 1×10 min).

Immunological Detection Kit (Boehringer Mannheim)

This protocol follows closely the one provided by the manufacturer, butwith minor modification. Each section is dipped briefly into buffer 1(100 mM maleic acid, 150 mM NaCl, adjusted to pH 7.5 [20° C.] with NaOH,autoclaved) for equilibration and the excess liquid drained off beforeaddition of 100 μl of buffer 2 (1% blocking reagent ) is added to covereach tissue section. This is incubated in a humid chamber at roomtemperature for 20 min. This is followed by incubation with 100 μl ofanti-DIG antibodies conjugated to alkaline phosphatase (1:500 v/v in 1%blocking reagent. Blocking reagent: a 100 mg/ml stock is prepared bydissolving the supplied powder form in buffer 1. This is then autoclavedand aliquoted into eppendorf tubes and frozen at −20° C. This stock isdiluted 1:10 in buffer 1 to make the 1% blocking reagent.) A coverslipis put over the section to prevent evaporation and to ensure that thetissue section is completely covered with the antibody conjugate. Thisis placed in a humid chamber for 1 hr at room temperature. Sections arethen incubated in buffer 1 for 20 min (2×5 min and 1×10 min) to wash offthe unbound antibodies and equilibrated for another 5 min in 37° C.buffer 3 (100 mM Tris HCl, 100 mM NaCl, 50 mM MgCl₂, adjusted to pH 9.5[20° C.] with NaOH). The colour-substrate stock solution (nitrobluetetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate [NBT/BCIP]) isdiluted 1:50 in 37° C. buffer 3 just prior to use. Detection buffer 3 isdrained off sections and 120 μl of the freshly prepared colour-substratesolution is distributed over the tissue to detect the hybridized probe.The sections are covered with coverslip each and left in a 37° C. dark,humid chamber for at least 1 hour. Color development is carefullymonitored until the reaction on the positive control slide isappropriate. Color development can range between 1 to 4 hours. Afterrinsing sections several times in 37° C. distilled water, they arestained with nuclear fast red for 1 to 5 s depending on the tissue size.Excess stain is rinsed off by several dips into distilled water andsections are mounted in a water-based mounting medium (DPX Mountant,BDH).

The system has been tested out on cytospin cells, colon, stomachsections that undergo PCRISH to (1) a reaction mixture without a probeand (2) a reaction mixture with a probe that has no complementarysequences in the tissue. There is no endogenous activity to giveartifacts.

TABLE 1 Primer Code Map Map Position Sequence 5′–3′ Gene ECM-246AE000201 246-266 atgactggtttagtaaaatgg cspG (SEQ ID NO. 1) ECM-850AE000201 850-830 tcaatattcactgttaacctc sfa (SEQ ID NO. 2) ECM-1163AE000201 1143-1163 cattgcgtaaccaatcaccgc yccM (SEQ ID NO. 3) ECM-1958AE000201 1958-1938 gcaagtagcacgacatttgtc yccM (SEQ ID NO. 4) torT-5129AE000201 5129-5148 ggtgcaagcctctacgccgc torT (SEQ ID NO. 5) torT-5750AE000201 5750-5731 tgccgcctctgccgcaatgg torT (SEQ ID NO. 6) torC-7218AE000201 7218-7242 aacttgccgagcgtgaatgggcgcg torC (SEQ ID NO. 7)torC-7761 AE000201 7761-7737 gtggcctgcaacttgctccactcgg torC (SEQ ID NO.8) torA-8332 AE000201 8332-8356 tatccgatggtacgcgtggactggc torA (SEQ IDNO. 9) torA-8891 AE000201 8891-8867 gcaatgtgcttcacatgctcgcgcc torA (SEQID NO. 10) torD-10574 AE000201 10574-10593 gaccacgctgacagcacaac torD(SEQ ID NO. 11) torD-11160 AE000201 11160-11141 ggtggtcgcactccactaactorD (SEQ ID NO. 12) CD-415 AE000202 415-436 gctttcccccaatctttacgtg cbpA(SEQ ID NO. 13) CD-1351 AE000202 1351-1329 gatttacgcgagataacgctatg cbpA(SEQ ID NO. 14) agp-3151 AE000202 3151-3172 cgctaatcgccgcagctgtggc agp(SEQ ID NO. 15) agp-4359 AE000202 4359-4336 cgctatcaaacttatccatcgggc agp(SEQ ID NO. 16) Wrb-4807 AE000202 4807-4836tgtgaaacgtcaaataattcctgcgctgcg wrbA (SEQ ID NO. 17) Wrb-5235 AE0002025235-5206 catgtacggacatattgaaacgatggcacg wrbA (SEQ ID NO. 18) ycdG-6073AE000202 6073-6094 ctcctgatgaacaacttctggc ycdG (SEQ ID NO. 19) 81B-7223AE000202 7223-7240 ggatccagccccatcaga ycdG (SEQ ID NO. 20) 81B-7278AE000202 7278-7297 cgtgttgaacgcccattact ycdG (SEQ ID NO. 21) ycdG-7359AE000202 7359-7340 tcgacctctacagagagcgg ycdG (SEQ ID NO. 22) OH-7419AE000202 7419-7436 acaagcagggcgcatcag b1007 (SEQ ID NO. 23) OH-7562AE000202 7562-7590 acgaaaccagagcctcttccagttgcggg b1007 (SEQ ID NO. 24)81B-7754 AE000202 7773-7754 gcccacattactggtgtgcc b1007 (SEQ ID NO. 25)81B-7794 AE000202 7794-7774 ctgcagtgtgaccgatacgcc b1007 (SEQ ID NO. 26)OH-7985 AE000202 7985-7966 atagcagcaagctttatgcg b1008 (SEQ ID NO. 27)New1-8160 AE000202 8160-8179 cggcaagttgtgggctggag b1008 (SEQ ID NO. 28)New1-9704 AE000202 9704-9684 cgtaattattcccgctggcag b1010 (SEQ ID NO. 29)New2-9731 AE000202 9731-9749 gcgatatgagcaaaggacg b1011 (SEQ ID NO. 30)B-11375 AE000202 11375-11356 ctgtcgatgatcaaactgcg b1012 (SEQ ID NO. 31)New2-503 AE000203 503-484 gcatctccatacagaacagg ycdc (SEQ ID NO. 32)putP-5944 AE000203 5944-5963 ctgggttacttcgggcagcc putP (SEQ ID NO. 33)putP-6693 AE000203 6693-6674 cggagccgaatgatagtgcg putP (SEQ ID NO. 34)HP-178 E25742 178-199 gctaagagatcagcctatgtcc HP16S (SEQ ID NO. 35) rRNAHP-228 E25742 228-252 accaaggctatgacgggtatccggc HP16S (SEQ ID NO. 36)rRNA HP-513 E25742 513-489 gcgctctttacgcccagtgattccg HP16S (SEQ ID NO.37) rRNA HP-775 E25742 775-751 gccctccaacaactagcatccatcg HP16S (SEQ IDNO. 36) rRNA

TABLE 2a Grid A lists the different types of bacteria genomic DNA loadedonto nylon plus membrane and hybridized to random primed³²P-radiolabeled gene probes. W X Y Z 1 Placenta Escherichia Salmonellavulneris choleraesuis 2 Helicobacter Hafnia Shigella flexneri TG2 pylorialvei 3 Aeromonas Klebsiella Serratia jandanei pneumoniae marcescens 4Citrobacter Morganella Shigella sonnei K12 freundii morganii 5 CedeceaProteus Yersinia lapagei vulgaris enterocolitica 6 EnterobacterProvidencia Escherichia 078: H11 cloacae alcalifaciens blattae 7Escherichia Pseudomonas Enterobacter hermannii aeruginosa agglomerans 8Edwardsiella Shigella boydii 219/1 sssDNA tarda Shigella sonnei Grid Blists the different types of bacteria genomic DNA loaded onto nylon plusmembrane and hybridized to random primed ³²P-radiolabeled gene probes. WX Y Z 1 Placenta Escherichia Salmonella 078: H11 vulneris choleraesuis 2Helicobacter Hafnia Shigella flexneri 142/31 pylori alvei 3 AeromonasKlebsiella Serratia K12 jandanei pneumoniae marcescens 4 CitrobacterMorganella Shigella sonnei TG2 freundii morganii 5 Cedecea ProteusYersinia 179/36 lapagei vulgaris enterocolitica 6 EnterobacterProvidencia Escherichia 197/5 cloacae alcalifaciens blattae 7Escherichia Pseudomonas Enterobacter 117/3B hermannii aeruginosaagglomerans 8 Edwardsiella Shigella boydii 219/1 sssDNA tarda Shigellasonnei Grid C lists the different types of bacteria genomic DNA loadedonto nylon plus membrane and hybridized to random primed³²P-radiolabeled gene probes. W X Y Z 1 Placenta Escherichia Salmonella078: H11 vulneris choleraesuis 2 Helicobacter Hafnia Shigella flexneri196/1 pylori alvei 3 Aeromonas Klebsiella Serratia K12 jandaneipneumoniae marcescens 4 Citrobacter Morganella Shigella sonnei TG2freundii morganii 5 Cedecea Proteus Yersinia 196/28 lapagei vulgarisenterocolitica 6 Enterobacter Providencia Escherichia 197/5 cloacaealcalifaciens blattae 7 Escherichia Pseudomonas Enterobacter 218/40hermannii aeruginosa agglomerans 8 Edwardsiella Shigella boydii 219/10157: H7 tarda Shigella sonnei Grid D lists the DNA of E. coli and grampositive bacteria isolated from patients' fecal specimen and loaded ontonylon plus membrane and hybridized to different random primed ³²Plabeled gene probes. A B C D E F 1 Placental 139/6 158/20 174/TM5 206/13HP 2 114/TB 142/31 158/37 179/36 218/40 114/3g 3 117/3B 145/34 159/193/6 224/1 115/TA TM17 4 119/1TM1 145/40 162/20 196/1 231/1 116/TC 5129/20 152/ 164/1 196/28 236/4 116/TD W2-35 6 135/35 154/1 168/4 197/5240/28 117/2D 7 130/22 154/9 168/38 205/T18 252/22 O157: H7 8 136/36156/ 172/33 205/T34 K12 Staphylococcus TM22 Grid E lists the range ofdifferent amount of Enterobacter cloacae and K12 E. coli DNA loaded ontonylon plus membrane and hybridized to different types of random primed³²P gene probe. Enterobacter E. coli E. coli Cloacae (ng) K12 (ng) K12(ng) 1 1 1 2 100 2 2 3 5 5 4 100 10 10 5 20 20 6 50 50 Grid F lists theH. pylori primer directed PCR amplified DNA product of H. pylori and E.coli DNA and total DNA of H. pylori and E. coli isolates obtained frompatients' fecal specimens. These are loaded onto nylon plus membrane andhybridized to random primed ³²P radiolabeled H. pylori ribosomal geneprobe (HP). A Volume of C Diluted Rows 1–5: (1:10) PCR PCR reaction Breaction mixture mixture after Rows 1–5: PCR reaction mixture Rows 6–8:E. coli amplification Rows 6–8: HP Genomic DNA Genomic DNA 1  1 μl 5 μlof HP diluted reaction mix 5 μl of K12 (HP)  5 μl of K12 reactionmixture reaction mixture 2  5 μl 5 μl of HP diluted reaction mix 5 μl of142/31 (HP) 5 μl of 142/31 reaction mixture reaction mixture 3 10 μl 5μl of HP diluted reaction mix 5 μl of 179/36 (HP) 5 μl of 179/36reaction mixture reaction mixture 4 50 μl 5 μl of HP diluted reactionmix 5 μl of 197/5 (HP) 5 μl of 197/5 reaction mixture reaction mixture 5 1 ul 5 μl of HP diluted reaction mix 5 μl of 117/3B (mixed 5 μl of117/3B reaction mixture reaction mixture E. coli isolates) 6  5 ul  10ng of HP  10 ng of 117/3B (mixed genomic DNA genomic DNA E. coliisolates) 7 10 ul  50 ng of HP  50 ng of 117/3B (mixed genomic DNAgenomic DNA E. coli isolates) 8 50 ul 200 ng of HP 200 ng of 117/3B(mixed genomic DNA genomic DNA E. coli isolates)

TABLE 2b Bacteria source. Bacterial genus Bacteria species SourceEscherichia 078: H11 ATCC 35401 (E. coli ) 078: K80: H12 ATCC 438960157: H7 ATCC 43895 029: NM ATCC 43892 0111 ATCC 33780 0142: K86(B) ATCC23985 TG2 gift K12 ATCC 29947 Escherichia E. hermannii ATCC 33650(non-coli) E. vulneris ATCC 33821 E. blattae ATCC 29907 Shigella S.flexneri ATCC 29903 (serotype 2A) S. sonnei ATCC 29930 S. boydii ATCC8700 (serotype 2) Edwardsiella E. tarda ATCC 15947 (01433: H1)Salmonella S. choleraesuis ATCC 43971 Citrobacter C. freundii ATCC 8090klebsiella K. pneumoniae ATCC 11296 (Ozaenae type4) Enterobacter E.cloacae ATCC 13047 Hafnia H. alvei ATCC 13337 Serratia S. marcescensATCC 13880 Proteus P. vulgaris ATCC 13315 Morganella M. morganii ATCC49948 Providencia P. alcalifaciens ATCC 9886 Yersinia Y. enterocoliticaATCC 29913 Cedecea C. lapagei ATCC 33432 Aeromonas A. jandanei ATCC49568 Enterobacter E. agglomerans field isolates Pseudomonas P.aeruginosa field isolates Helicobacter H. pylori field isolates HumanPlacental DNA Sigma (UK) Salmon Salmon Sigma (UK) sperm DNA TG2: GibsonTJ: Studies on the Epstein-Bar-Virus Genome, Ph.D Thesis 1984. CambridgeUniversity, UK.

TABLE 3 Result of in-vitro simulation of PCRISH. b1007/b1008 ycdG/b1007genes genes HP gene PCR Probe PCR Probe PCR Probe primers primersprimers primers primers primers Bacterial Bacteria OH-7419 OH-756281B-7223 81B-7278 HP-178 HP-228 genus species OH-7985 81B-7794 81B-779481B-7754 HP-775 HP-513 Escherichia 078: H11 + + + + − − (E. coli)TG2 + + + + − − K12 + + + + − − 142/31 + + + + − − 179/36 + + + + − −197/5 + + + + − − Escherichia E. hermannii − − − − − − (non-coli) E.vulneris + − − − − − E. blattae − − − − − − Shigella S. flexneri + + + +− − (serotype 2A) S. sonnei + + + + − − S. sonnei: + + + + − − 219/1 S.boydii − − − − − − (serotype 2) Edwardsiella E. tarda − − − − − −(01433: H1) Salmonella S. choleraesuis + − − − − − Citrobacter C.freundii − − − − − − Klebsiella K. pneumoniae − − − − − − (Ozaenaetype4) Enterobacter E. cloacae − − − − − − Hafnia H. alvei − − − − − −Proteus P. vulgaris − − − − − − Morganella M. morganii − − − − − −Providencia P. alcalifaciens + − − − − − Yersinia Y. − − − − − −enterocolitica Cedecea C. lapagei + − − − − − Aeromonas A. jandanei − −− − − − Enterobacter E. agglomerans − − − − − − Pseudomonas P.aeruginosa − − − − − − Helicobacter H. pylori − − − − + + HumanPlacental DNA − − − − − − Salmon Salmon − − − − − − sperm DNA

1. A probe nucleotide sequence which is specific to E. coli- and/orShigella species or related microorganism, the nucleotide sequenceconsisting of a sequence from SEQ ID NO: 40, the sequence from SEQ IDNO: 40 comprising at least one of the sequence consisting of nucleotides415 to 1351, the sequence consisting of nucleotides 3151 to 4359, thesequence consisting of nucleotides 4807 to 5235, the sequence consistingof nucleotides 6073 to 7359, the sequence consisting of nucleotides 7223to 7794, the sequence consisting of nucleotides 7278 to 7773, thesequence consisting of nucleotides 7419 to 7985, the sequence consistingof nucleotides 7562 to 7794, the sequence consisting of nucleotides 8160to 9704 or the sequence consisting of nucleotides 9731 to 11375, andpresent mainly in the nucleus of cancer cells and in the normal cellsadjacent to cancer cells, for the identification of a gastrointestinalcancer or tumour or a predisposition to same.
 2. A method for detectingthe presence of E. coli or Shigella species or related microorganisms ina sample, said method comprising subjecting said sample to geneticanalysis using an E. coli- or Shigella species-specific nucleotidesequence consisting of a sequence from SEQ ID NO: 40, the sequence fromSEQ ID NO: 40 comprising at least one of the sequence consisting ofnucleotides 415 to 1351, the sequence consisting of nucleotides 3151 to4359, the sequence consisting of nucleotides 4807 to 5235, the sequenceconsisting of nucleotides 6073 to 7359, the sequence consisting ofnucleotides 7223 to 7794, the sequence consisting of nucleotides 7278 to7773, the sequence encompassed by nucleotides 7419 to 7985, the sequenceconsisting of nucleotides 7562 to 7794, the sequence consisting ofnucleotides 8160 to 9704 or the sequence consisting of nucleotides 9731to
 11375. 3. The method according to claim 2 wherein said geneticanalysis comprises amplification of nucleotide sequence present in thesample.
 4. The method according claim 2 wherein said E. coli- and/orShigella species-specific nucleotide sequence is labeled to provide anidentifiable signal.
 5. The method according to 2 wherein the samplecomprises a nucleic acid preparation from food, water, semi-solids orsemi-liquid material, mammalian tissue, or extract or cells thereof or anucleic acid preparation from said tissue, extract or cells.
 6. Themethod according to claim 5 wherein the sample is mammalian tissue orextract or cells thereof.
 7. The method according to claim 6 wherein thetissue, extract or cells are from a patient suffering from cancer orcellular instability or gastrointestinal infection, or a patient at riskof cancer or cellular instability.
 8. The method according to claim 7wherein the cancer is gastrointestinal cancer.
 9. The method accordingto claim 7 wherein the cancer is colon cancer.
 10. The method accordingto claim 7 wherein the cancer is stomach cancer.
 11. The methodaccording to claim 7 wherein the cancer is colorectal cancer.
 12. Anisolated nucleic acid molecule comprising the probe nucleotide sequenceof claim 1 wherein said probe nucleotide sequence is capable ofspecifically hybridizing to E. coli- and/or Shigella species'-derivednucleic acid molecules.
 13. A method of testing and selecting sequencesspecific to E. coli or Shigella species or related microorganisms in asample, said method comprising subjecting a nucleic acid moleculepreparation from said sample to genetic analysis using one or more E.coli or Shigella species'-specific nucleotide sequences consistig of asequence from SEQ ID NO: 40, the sequence from SEQ ID NO: 40 comprisingat least one of the sequence consisting of nucleotides 415 to 1351, thesequence consisting of nucleotides 3151 to 4359, the sequence consistingof nucleotides 4807 to 5235, the sequence consisting of nucleotides 6073to 7359, the sequence consisting of nucleotides 7223 to 7794, thesequence consisting of nucleotides 7278 to 7773, the sequence consistingof nucleotides 7419 to 7985, the sequence consisting of nucleotides 7562to 7794, the sequence consisting of nucleotides 8160 to 9704 or thesequence consisting of nucleotides 9731 to
 11375. 14. The methodaccording to claim 13 wherein said genetic analysis comprisesamplification of nucleotide sequence present in the sample.
 15. Themethod according to claim 13 wherein said E. coli- and/or Shigellaspecies-specific nucleotide sequence is labeled to provide anidentifiable signal.
 16. The method according to claim 13 wherein thesample comprises mammalian tissue or extract or cells thereof.
 17. Themethod according to claim 16 wherein the tissue, extract or cells arefrom a patient suffering from cancer or cellular instability orgastrointestinal infection, or a patient at risk of cancer or cellularinstability.
 18. The method according to claim 17 wherein the cancer isgastrointestinal cancer.
 19. The method according to claim 17 whereinthe cancer is colon cancer.
 20. The method according to claim 17 whereinthe cancer is stomach cancer.
 21. The method according to claim 17wherein the cancer is colorectal cancer.
 22. A nucleotide sequenceidentified by the method according to claim 13 wherein said sequence iscapable of specifically hybridizing to E. coli and/or Shigellaspecies'-derived nucleic acid molecules.